Replicative minicircle vectors with improved expression

ABSTRACT

The present invention relates to the production and use of covalently closed circular (ccc) recombinant DNA molecules such as plasmids, cosmids, bacterial artificial chromosomes (BACs), bacteriophages, viral vectors and hybrids thereof, and more particularly to vector modifications that improve expression of said DNA molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application Ser. No. 61/796,765, entitled “Replicative Minicircle vectors with improved expression” which was filed Nov. 19, 2012, the entire contents of which are incorporated herein by reference and to U.S. Provisional Patent Application Ser. No. 61/743,219, entitled “DNA Plasmids With Improved Expression” which was filed Aug. 29, 2012, the entire contents of which are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was supported in part with government support under Grant No. R44GM080768, awarded by the National Institutes of Health. The government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to a family of eukaryotic expression plasmids useful for gene therapy, obtaining improved genetic immunization, natural interferon production, and more particularly, for improving the expression of plasmid encoded antigens or therapeutic genes.

Such recombinant DNA molecules are useful in biotechnology, transgenic organisms, gene therapy, therapeutic vaccination, agriculture and DNA vaccines.

BACKGROUND OF THE INVENTION

E. coli plasmids have long been an important source of recombinant DNA molecules used by researchers and by industry. Today, plasmid DNA is becoming increasingly important as the next generation of biotechnology products (e.g. gene medicines and DNA vaccines) make their way into clinical trials, and eventually into the pharmaceutical marketplace. Plasmid DNA vaccines may find application as preventive vaccines for viral, bacterial, or parasitic diseases; immunizing agents for the preparation of hyper immune globulin products; therapeutic vaccines for infectious diseases; or as cancer vaccines. Plasmids are also utilized in gene therapy or gene replacement applications, wherein the desired gene product is expressed from the plasmid after administration to the patient.

Therapeutic plasmids often contain a pMB1, ColEl or pBR322 derived replication origin. Common high copy number derivatives have mutations affecting copy number regulation, such as ROP (Repressor of primer gene) deletion, with a second site mutation that increases copy number (e.g. pMB 1 pUC G to A point mutation, or ColE1 pMM1). Higher temperature (42° C.) can be employed to induce selective plasmid amplification with pUC and pMM 1 replication origins.

U.S. Pat. No. 7,943,377 (Carnes, A E and Williams, J A, 2011) disclose methods for fed-batch fermentation, in which plasmid-containing E. coli cells were grown at a reduced temperature during part of the fed-batch phase, during which growth rate was restricted, followed by a temperature up-shift and continued growth at elevated temperature in order to accumulate plasmid; the temperature shift at restricted growth rate improved plasmid yield and purity. Other fermentation processes for plasmid production are described in Carnes A. E. 2005 BioProcess Intl 3:36-44, which is incorporated herein by reference in its entirety.

The art teaches that one of the limitations of application of plasmid therapies and plasmid vaccines is regulatory agency (e.g. Food and Drug Administration, European Medicines Agency) safety concerns regarding 1) plasmid transfer and replication in endogenous bacterial flora, or 2) plasmid encoded selection marker expression in human cells, or endogenous bacterial flora. Additionally, regulatory agency guidance's recommend removal of all non essential sequences in a vector. Plasmids containing a pMB1, ColE1 or pBR322 derived replication origin can replicate promiscuously in E. coli hosts. This presents a safety concern that a plasmid therapeutic gene or antigen will be transferred and replicated to a patient's endogenous flora. Ideally, a therapeutic or vaccine plasmid would be replication incompetent in endogenous E. coli strains. This requires replacement of the pMB1, ColE1 or pBR322 derived replication origin with a conditional replication origin that requires a specialized cell line for propagation. As well, regulatory agencies such as the EMEA and FDA are concerned with utilization of antibiotic resistance or alternative protein markers in gene therapy and gene vaccine vectors, due to concerns that the gene (antibiotic resistance marker or protein marker) may be expressed in a patients cells. Ideally, plasmid therapies and plasmid vaccines would be 1) replication incompetent in endogenous E. coli strains, 2) would not encode a protein based selection marker and 3) be minimalized to eliminate all non essential sequences.

The art further teaches that one of the limitations of application of plasmid therapies and vaccines is that transgene expression is generally very low. Vector modifications that improve antigen expression (e.g. codon optimization of the gene, inclusion of an intron, use of the strong constitutive CMV or CAGG promoters versus weaker or cell line specific promoter) are highly correlative with improved in vivo expression and, where applicable, immune responses (reviewed in Manoj S, Babiuk L A, van Drunen Little-van den Hurk S. 2004 Crit Rev Clin Lab Sci 41: 1-39). A hybrid CMV promoter (CMV/R), which increased antigen expression, also improved cellular immune responses to HIV DNA vaccines in mice and nonhuman primates (Barouch DH, Yang ZY, Kong WP, Korioth-Schmitz B, Sumida S M, Truitt D M, Kishko M G, Arthur J C, Miura A, Mascola J R, Letvin N L, Nabel G J. 2005 J Virol. 79: 8828-8834). A plasmid containing the woodchuck hepatitis virus posttranscriptional regulatory element (a 600 by element that increases stability and extranuclear transport of RNA resulting in enhanced levels of mRNA for translation) enhanced antigen expression and protective immunity to influenza hemagglutinin (HA) in mice (Garg S, Oran A E, Hon H, Jacob J. 2004 J Immunol. 173: 550-558). These studies teach that improvement in expression beyond that of current CMV based vectors may generally improve immunogenicity and, in the case of gene therapeutics, efficacy.

Transgene expression duration from plasmid vectors is reduced due to promoter inactivation mediated by the bacterial region (i.e. region encoding bacterial replication origin and selectable marker which is encoded in the spacer region) of the vector (Chen Z Y, He C Y, Meuse L, Kay M A. 2004. Gene Ther 11:856-864; Suzuki M, Kasai K, Saeki Y. 2006. J Virol 80:3293-3300). This results in short duration transgene expression. A strategy to improve transgene expression duration is to remove the bacterial region of the plasmid. For example, minicircle and ‘linear Minimalistic immunogenic defined gene expression’ (MIDGE) vectors have been developed which do not contain a bacterial region. Removal of the bacterial region in minicircle vectors improved transgene expression duration (Chen et al Supra, 2004). In minicircle vectors, the eukaryotic region polyadenylation signal is covalently linked to the eukaryotic region promoter. This linkage (spacer region) can tolerate a spacer sequence of at least 500 by since in vivo expression duration is improved with plasmid vectors in which the bacterial region is removed or replaced with a spacer sequence (spacer region) up to 500 by in length (Lu J, Zhang F, Xu S, Fire A Z, Kay M A. 2012. Mol Ther. 20:2111-9).

However, methods to manufacture MIDGE and minicircle vectors are expensive and not easily scalable. Creating terminal loops on MIDGE vectors in vitro is problematic, requiring in vitro ligation of annealed primers to restriction digested vector. For minicircle vectors, E. coli based manufacturing systems have been developed in which, after plasmid production, the bacterial region and the eukaryotic region are separated and circularized into minicircles via the action of phage recombinases on recognition sequences in the plasmid. In some methods, a restriction enzyme is then utilized to digest the bacterial minicircle at a unique site to eliminate this difficult to remove contaminant. These production procedures are very inefficient. For example, optimal manufacture of minicircle vectors yields only 5 mg of minicircle per liter culture (Kay M A, He C Y, Chen Z Y. 2010. Nat Biotechnol 28:1287-1289).

A solution is needed to develop eukaryotic expression vectors that contain short spacer regions preferably 500 by or less that can be efficiently manufactured. These vectors should not encode a protein based selection marker and should be minimalized to eliminate all non essential sequences.

SUMMARY OF THE INVENTION

The present invention relates to a family of minimalized eukaryotic expression plasmids with short spacer regions that preferably are replication incompetent in endogenous flora and surprisingly have dramatically improved in vivo expression. These vectors are useful for gene therapy, genetic immunization and or interferon therapy.

Improved vectors that utilize novel intronic bacterial regions in which bacterial propagation and optionally selection functions are nested within the eukaryotic expression cassette are disclosed.

One object of the invention is to provide improved expression plasmid vectors.

Another object of the invention is to provide eukaryotic expression vectors containing short spacer regions that may be efficiently manufactured.

According to one object of the invention, a method of improving expression from an expression plasmid vector comprises modifying the plasmid DNA to replace the spacer region encoded pMB1, Co1E1, pBR322, R6K, ColE2-P9 or ColE2-P9 related derived replication origin with an alternative intronic replication origin selected from the group consisting of an R6K gamma replication origin, a ColE2-P9 replication origin, a ColE2-P9 related replication origin, a pUC replication origin, a P_(min) pUC replication origin; transforming the modified plasmid DNA into a bacterial cell line rendered competent for transformation; and isolating the resultant transformed bacterial cells.

According to one object of the invention, a composition for construction of a short spacer region eukaryotic expression vector with high yield manufacture comprises an R6K origin with at least 90% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 11 and SEQ ID NO: 12, and a plasmid DNA encoded eukaryotic region, wherein said R6K origin is operably linked to an intron within said plasmid DNA eukaryotic region. According to still another object of the invention, a RNA selection marker is incorporated into the vector adjacent to the R6K replication origin. According to still another object of the invention, a RNA selection marker is incorporated into the vector within a second intron or within the spacer region. According to still another object of the invention, the RNA selection marker is selected from the group consisting of: an RNA-OUT selection marker that encodes an RNA-IN regulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO: 21; an RNAI selection marker that encodes an RNAII regulating RNAI RNA with at least 95% sequence identity to SEQ ID NO: 33; an IncB RNAI selection marker encoding an RNAII regulating RNAI RNA with at least 95% sequence identity to SEQ ID NO: 35. According to still another object of the invention, a RNA-OUT selection marker selected from the group consisting of: SEQ ID NO: 20 and SEQ ID NO: 22 is incorporated into the vector adjacent to the R6K origin. According to still another object of the invention, a RNA-OUT selection marker selected from the group consisting of: SEQ ID NO: 20 and SEQ ID NO: 22 is incorporated into the vector within a second intron. According to still another object of the invention, a RNA-OUT selection marker selected from the group consisting of: SEQ ID NO: 20 and SEQ ID NO: 22 is incorporated into the vector within the spacer region. According to still another object of the invention, the RNA-OUT selection marker -R6K origin operably linked to the intron is selected from the group consisting of: SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28. According to another object of the invention, said intronic R6K origin improves said vector expression compared to a corresponding vector containing a pMB1, ColE1 or pBR322 derived replication origin encoded in the backbone spacer region. According to still another object of the invention, said eukaryotic region has at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 30, SEQ ID NO: 31.

According to one object of the invention, a composition for construction of a short spacer region eukaryotic expression vector with high yield manufacture comprises a ColE2-P9 origin with at least 90% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 13, 14, 15, or 16, and a plasmid DNA encoded eukaryotic region, wherein said ColE2-P9 origin is operably linked to an intron within said plasmid DNA encoded eukaryotic region. According to still another object of the invention, a primosomal assembly site (ssiA) is optionally incorporated into the vector adjacent to the ColE2-P9 origin. According to still another object of the invention, a RNA selection marker is incorporated into the vector adjacent to the ColE2-P9 replication origin. According to still another object of the invention, a RNA selection marker is incorporated into the vector within a second intron or within the spacer region. According to still another object of the invention, the RNA selection marker is selected from the group consisting of: an RNA-OUT selection marker that encodes an RNA-IN regulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO: 21; an RNAI selection marker that encodes an RNAII regulating RNAI RNA with at least 95% sequence identity to SEQ ID NO: 33; an IncB RNAI selection marker encoding an RNAII regulating RNAI RNA with at least 95% sequence identity to SEQ ID NO: 35. According to still another object of the invention, a RNA-OUT selection marker selected from the group consisting of: SEQ ID NO: 20 and SEQ ID NO: 22 is incorporated into the vector adjacent to the ColE2-P9 origin. According to still another object of the invention, a RNA-OUT selection marker selected from the group consisting of SEQ ID NO: 20 and SEQ ID NO: 22 is incorporated into the vector within a second intron. According to still another object of the invention, a RNA-OUT selection marker selected from the group consisting of: SEQ ID NO: 20 and SEQ ID NO: 22 is incorporated into the vector within the spacer region. According to still another object of the invention, the RNA-OUT selection marker -ColE2-P9 origin operably linked to the intron is selected from the group consisting of SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25. According to another object of the invention, said intronic ColE2-P9 origin improves said vector expression compared to a corresponding vector containing a pMB1, ColE1 or pBR322 derived replication origin encoded in the backbone spacer region. According to still another object of the invention, said eukaryotic region has at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 30, SEQ ID NO: 31.

According to one object of the invention, a composition for construction of a short spacer region eukaryotic expression vector with high yield manufacture comprises a pUC origin, and a plasmid DNA encoded eukaryotic region, wherein said pUC origin is operably linked to an intron within said plasmid DNA eukaryotic region. According to still another object of the invention, a RNA selection marker is incorporated into the vector adjacent to the pUC replication origin. According to still another object of the invention, a RNA selection marker is incorporated into the vector within a second intron or within the spacer region. According to still another object of the invention, the RNA selection marker is selected from the group consisting of an RNA-OUT selection marker that encodes an RNA-IN regulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO: 21; an RNAI selection marker that encodes an RNAII regulating RNAI RNA with at least 95% sequence identity to SEQ ID NO: 33; an IncB RNAI selection marker encoding an RNAII regulating RNAI RNA with at least 95% sequence identity to SEQ ID NO: 35. According to still another object of the invention, a RNA-OUT selection marker selected from the group consisting of: SEQ ID NO: 20 and SEQ ID NO: 22 is incorporated into the vector adjacent to the pUC origin. According to still another object of the invention, a RNA-OUT selection marker selected from the group consisting of: SEQ ID NO: 20 and SEQ ID NO: 22 is incorporated into the vector within a second intron. According to still another object of the invention, a RNA-OUT selection marker selected from the group consisting of: SEQ ID NO: 20 and SEQ ID NO: 22 is incorporated into the vector within the spacer region. According to still another object of the invention, the RNA-OUT selection marker -pUC origin operably linked to the intron is SEQ ID NO: 29. According to another object of the invention, said intronic pUC origin improves said vector expression compared to a corresponding vector containing a pMB1, ColE1 or pBR322 derived replication origin encoded in the backbone spacer region. According to still another object of the invention, said eukaryotic region has at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 30, SEQ ID NO: 31.

According to one object of the invention, a composition for construction of a short spacer region eukaryotic expression vector with high yield manufacture comprises a P_(min) pUC origin with at least 90% sequence identity to SEQ ID NO: 38, and a plasmid DNA encoded eukaryotic region, wherein said P_(min), pUC origin is operably linked to an intron within said plasmid DNA eukaryotic region. According to still another object of the invention, a RNA selection marker is incorporated into the vector adjacent to the P_(mm) pUC replication origin. According to still another object of the invention, a RNA selection marker is incorporated into the vector within a second intron or within the spacer region. According to still another object of the invention, the RNA selection marker is selected from the group consisting of: an RNA-OUT selection marker that encodes an RNA-IN regulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO: 21; an RNAI selection marker that encodes an RNAII regulating RNAI RNA with at least 95% sequence identity to SEQ ID NO: 33; an IncB RNAI selection marker encoding an RNAII regulating RNAI RNA with at least 95% sequence identity to SEQ ID NO: 35. According to still another object of the invention, a RNA-OUT selection marker selected from the group consisting of: SEQ ID NO: 20 and SEQ ID NO: 22 is incorporated into the vector adjacent to the P_(mm) pUC origin. According to still another object of the invention, a RNA-OUT selection marker selected from the group consisting of: SEQ ID NO: 20 and SEQ ID NO: 22 is incorporated into the vector within a second intron. According to still another object of the invention, a RNA-OUT selection marker selected from the group consisting of: SEQ ID NO: 20 and SEQ ID NO: 22 is incorporated into the vector within the spacer region. According to still another object of the invention, the RNA-OUT selection marker -P_(min) pUC origin operably linked to the intron is SEQ ID NO: 39. According to another object of the invention, said intronic P_(min) pUC origin improves said vector expression compared to a corresponding vector containing a pMB1, ColE1 or pBR322 derived replication origin encoded in the backbone spacer region. According to still another object of the invention, said eukaryotic region has at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 30, SEQ ID NO: 31.

Further objects and advantages of the invention will become apparent from a consideration of the drawings and ensuing description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the NTC8485 pUC origin expression vector;

FIG. 2 depicts bioinformatics analysis of an intron containing the gWIZ bacterial region (GBR) encoded kanR selection marker-pUC origin;

FIG. 3 depicts bioinformatics analysis of introns containing the NTC9385P2 bacterial region (P2) encoded RNA-OUT selection marker-pUC origin in both orientations;

FIG. 4 depicts the NTC9385P2a-01-EGFP and NTC9385P2a-02-EGFP intronic pUC origin-RNA-OUT replicative minicircle expression vectors;

FIG. 5 shows plasmid quality from intronic pUC origin-RNA-OUT expression vectors NTC9385P2a-O1-EGFP, NTC9385P2a-02-EGFP, NTC9385P2-01-EGFP and NTC9385P2-02-EGFP vectors versus a comparator backbone spacer region encoded pUC origin-RNA-OUT expression vector NTC8385-EGFP;

FIG. 6 depicts the NTC9385R2a-O1-EGFP and NTC9385R2a-O2-EGFP intronic R6K origin-RNA-OUT replicative minicircle expression vectors;

FIG. 7 depicts the NTC9385C2a-O1-EGFP and NTC9385C2a-O2-EGFP intronic ColE2 origin-RNA-OUT replicative minicircle expression vectors;

FIG. 8 shows plasmid quality from Table 6 fermentations of intronic R6K origin-RNA-OUT expression vectors NTC9385R2-O1-EGFP, NTC9385R2-O2-EGFP, NTC9385R2a-O1-EGFP and NTC9385R2a-O2-EGFP vectors, versus a comparator backbone spacer region encoded R6K origin-RNA-OUT expression vector NTC9385R-EGFP;

FIG. 9 depicts the NTC9385R2b-02-EGFP intronic R6K origin- spacer region RNA-OUT replicative minicircle expression vectors;

FIG. 10 depicts the pMB1 and ColE1 RNA I antisense repressor RNA; and

FIG. 11 depicts an IncB RNAI based RNA selectable marker.

Table 1: Intron encoded RNA-OUT selection/replication origin does not prevent transgene expression; Table 2: Improved expression with intron encoded RNA-OUT selection/replication origin; Table 3: Intron functional analysis—Splicing accuracy and export efficiency; Table 4: Intron vector expression efficiency; Table 5: Replicative minicircle vector expression in vitro (lipofectamine) and in vivo (intradermal delivery with electroporation); Table 6: Intronic RNA-OUT AF selection plasmid fermentation yields; Table 7: High level expression with vectors with pMB1 RNAI encoded in the spacer region or intron; Table 8: Accurate splicing with replicative minicircle vectors with pMB1 RNAI and minimal pUC origin encoded in the intron; and Table 9: Robust expression with P2 (0.85) replicative minicircles.

SEQ ID NO:1: HTLV-IR-Rabbit β globin hybrid intron SEQ ID NO:2: HTLV-IR CMV hybrid intron SEQ ID NO:3: CMV intron SEQ ID NO:4: CpG free intron I 140 SEQ ID NO:5: Human β globin Murine IgG chimeric intron SEQ ID NO:6: Adenovirus leader—Murine IgG chimeric intron SEQ ID NO:7: Rabbit β globin intron SEQ ID NO:8: Truncated CMV intron SEQ ID NO:9: CAG (Chicken β Actin-rabbit β globin) intron SEQ ID NO:10: CMV-Rabbit β globin hybrid intron SEQ ID NO:11: R6K gamma origin SEQ ID NO:12: CpG free R6K gamma origin SEQ ID NO:13: ColE2 Origin (+7) SEQ ID NO:14: ColE2 Origin (Min) SEQ ID NO:15: ColE2 origin (Core) SEQ ID NO:16: CpG free ColE2 Origin (+7, CpG free) SEQ ID NO:17: CpG free ssiA [from plasmid R6K] SEQ ID NO:18: +7(CpG free) ColE2 origin-CpG free ssiA SEQ ID NO:19: +7(CpG free) ColE2 origin-CpG free ssiA- flanked by SphI and KpnI restriction sites SEQ ID NO:20: RNA-OUT Selectable Marker SEQ ID NO:21: RNA-OUT antisense repressor RNA SEQ ID NO:22: CpG free RNA-OUT selection marker. Flanked by KpnI (GGTACC) and BglII-EcoRI restriction sites SEQ ID NO:23: RNA-OUT-ColE2 origin bacterial region. [NheI site-ssiA-ColE2 Origin (+7)-RNA-OUT-KpnI site] SEQ ID NO:24: NTC9385C2 and NTC9385C2a intronic bacterial region. [filled NheI site-ssiA-ColE2 Origin (+7)-RNA-OUT-chewed KpnI site] Sequence show is O1; O2 is reverse complement SEQ ID NO:25: CpG free ColE2 RNA-OUT bacterial region. (CpG free ssiA-CpG free ColE2 origin-CpG free RNA-OUT selection marker)—flanked by EcoRI-SphI and BglII-EcoRI restriction sites SEQ ID NO:26: RNA-OUT-R6K gamma origin bacterial region. [NheI site-trpA terminator-R6K Origin-RNA-OUT-KpnI site] SEQ ID NO:27: NTC9385R2 and NTC9385R2a intronic R6K gamma origin-RNA-OUT bacterial region. [filled NheI site-trpA terminator-R6K Origin-RNA-OUT-chewed KpnI site] Sequence show is O1; O2 is reverse complement SEQ ID NO:28: CpG free R6K gamma origin RNA-OUT bacterial region. Flanked by EcoRI-SphI and BgilI-EcoRI restriction sites SEQ ID NO:29: NTC9385P2 and NTC9385P2a intronic pUC origin-RNA-OUT Bacterial region. [filled NheI site-trpA terminator-pUC Origin-RNA-OUT-chewed KpnI site] Sequence show is O1; O2 is reverse complement SEQ ID NO:30: NTC9385C2, NTC9385R2, NTC9385P2, NTC9385P2(0.85) Eukaryotic region. Bp 1 is start of CMV enhancer, by 1196 is end of polyadenylation site. Exon 2 encoded Sall (GTCGAC) and BglII (AGATCT) transgene cloning sites. Intron encoded HpaI (GTTAAC) bacterial region cloning site SEQ ID NO:31: NTC9385C2a, NTC9385R2a, NTC9385P2a and NTC9385P2a(0.85) Eukaryotic region. Bp 1 is start of CMV enhancer encoded boundary region, by 1292 is end of polyadenylation site. Exon 2 encoded Sail (GTCGAC) and BglII (AGATCT) transgene cloning sites. Intron encoded HpaI (GTTAAC) bacterial region cloning site SEQ ID NO:32: CpG free HTLV-IR-Rabbit β globin hybrid intron SEQ ID NO:33: RNAI antisense repressor RNA (pMB 1 plasmid origin RNAII antisense partner) SEQ ID NO:34: RNAI selectable Marker SEQ ID NO:35: IncB RNAI antisense repressor RNA (IncB plasmid origin RNAII antisense partner) SEQ ID NO:36: IncB RNAI selectable Marker, RNAI RNA (Sense strand) underlined. Flanked by DraIII-KpnI restriction sites SEQ ID NO:37: IncB RNAII-SacB. PstI-MamI restriction fragment SEQ ID NO:38: P_(min) pUC replication origin (minimal) SEQ ID NO:39: NTC9385P2(0.85) and NTC9385P2a(0.85) intronic pUC (0.85) Bacterial region [filled NheI site-trpA terminator-P_(min) pUC replication origin-RNA-OUT-chewed KpnI site] Sequence shown is O1; O2 is reverse

Definition of Terms A₄₀₅: Absorbance at 405 nanometers AF: Antibiotic-free APC: Antigen Processing Cell, for example, langerhans cells, plasmacytoid or conventional dendritic cells Approximately: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is the same or similar to a stated reference value BAC: Bacterial artificial chromosome Bacterial region: Region of a plasmid vector required for propagation and selection in the bacterial host BE: Boundary element: Eukaryotic sequence that that blocks the interaction between enhancers and promoters. Also referred to as insulator element. An example is the AT-rich unique region upstream of the CMV enhancer (XbaI to Spel region; FIG. 1) that can function as an insulator/boundary element (Angulo A, Kerry D, Huang H, Borst EM, Razinsky A, Wu J et al. 2000 J Viral 74: 2826-2839) bp: basepairs ccc: Covalently Closed Circular cI: Lambda repressor cITs857: Lambda repressor further incorporating a C to T (Ala to Thr) mutation that confers temperature sensitivity. cITs857 is a functional repressor at 28-30° C., but is mostly inactive at 37-42° C. Also called cI857 Cm^(R): Chloramphenicol resistance cmv: Cytomegalovirus CMV promoter boundary element: AT-rich region of the human cytomegalovirus (CMV) genome between the UL127 open reading frame and the major immediate-early (MIE) enhancer. Also referred to as unique region (Angulo et al. Supra, 2000) ColE2-P9 replication origin: a region which is specifically recognized by the plasmid-specified Rep protein to initiate DNA replication. Includes but not limited to ColE2-P9 replication origin sequences disclosed in SEQ ID NO:13: Co1E2 Origin (+7), SEQ ID NO:16: ColE2 Origin (+7, CpG free), SEQ ID NO:14: ColE2 Origin (Min) and SEQ ID NO:15: ColE2 Origin (core) and replication functional mutations as disclosed in Yagura et al 2006, J Bacteriol 188:999 included herein by reference ColE2 related replication origin: The ColE2-P9 origin is highly conserved across the ColE2-related plasmid family. Fifteen ColE2 related plasmid members including ColE3 are compared in Hiraga et al 1994, J Bacteriol. 176:7233 and 53 CoIE2 related plasmid members including ColE3 are compared in Yagura et al Supra, 2006. These sequences are included herein by reference ColE2-P9 plasmid: a circular duplex DNA molecule of about 7 kb that is maintained at about 10 to 15 copies per host chromosome. The plasmid encodes an initiator protein (Rep protein), which is the only plasmid-specified trans-acting factor essential for ColE2-P9 plasmid replication ColE2-P9 replication origin RNA-OUT bacterial region: Contains a ColE2-P9 replication origin for propagation and the RNA-OUT selection marker (e.g. SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25). Optionally includes a PAS, for example, the R6K plasmid CpG free ssiA primosomal assembly site (SEQ ID NO:17) or alternative ØX174 type or ABC type primosomal assembly sites, such as those disclosed in Nomura et al 1991 Gene 108:15 CoIE2 plasmid: NTC9385C, NTC9685C, NTC9385C2-O1, NTC9385C2-O2, NTC9385C2a-O1 NTC9385C2a-O2 vectors, as well as modifications and alternative vectors containing a ColE2-P9 replication origin that were disclosed in provisional patent application Ser. No. 61/743,219 entitled ‘DNA plasmids with improved expression’ (Docket DPWIE082912) and included herein by reference delivery methods: Methods to deliver gene vectors [e.g. poly(lactide-co-glycolide) (PLGA), ISCOMs, liposomes, niosomes, virosomes, chitosan, and other biodegradable polymers, electroporation, piezoelectric permeabilization, sonoporation, ultrasound, corona plasma, plasma facilitated delivery, tissue tolerable plasma, laser microporation, shock wave energy, magnetic fields, contactless magneto-permeabilization, gene gun, microneedles, microdermabrasion, topical DNA application, naked DNA injection, hydrodynamic delivery, high pressure tail vein injection, needle free biojector, liposomes, microparticles, microspheres, nanoparticles, virosomes, bacterial ghosts, bacteria, attenuated bacteria, etc] as known in the art and included herein by reference DNA replicon: A genetic element that can replicate under its own control; examples include plasmids, cosmids, bacterial artificial chromosomes (BACs), bacteriophages, viral vectors and hybrids thereof E. coli: Escherichia coli, a gram negative bacteria EGFP: Enhanced green fluorescent protein EP: Electroporation Eukaryotic expression vector: A vector for expression of mRNA, protein antigens, protein therapeutics, shRNA, RNA or microRNA genes in a target organism Eukaryotic region: The region of a plasmid that encodes eukaryotic sequences and/or sequences required for plasmid function in the target organism. This includes the region of a plasmid vector required for expression of one or more transgenes in the target organism including RNA Pol II enhancers, promoters, transgenes and polyA sequences. Additional functional eukaryotic region sequences include RNA Pol I or RNA Pol III promoters, RNA Pol I or RNA Pol HI expressed transgenes or RNAs, transcriptional terminators, S/MARs, boundary elements, etc FU: Fluorescence units g: Gram, kg for kilogram Hr(s): Hour(s) HTLV-I R: HTLV-I R 5′ untranslated region (UTR). Sequences and compositions were disclosed in Williams, J A 2008 World Patent Application WO2008153733 and included herein by reference IM: Intramuscular immune response: Antigen reactive cellular (e.g. antigen reactive T cells) or antibody (e.g. antigen reactive IgG) responses IncB RNAI: plasmid pMU720 origin encoded RNAI (SEQ ID NO: 35) that represses RNA II regulated targets (Wilson I W, Siemering K R, Praszkier J, Pittard A J. 1997. J Bacteriol 179:742) kan: Kanamycin kanR: Kanamycin Resistance gene Kd: Kilodalton kozak sequence: Optimized sequence of consensus DNA sequence gccRccATG (R=G or A) immediately upstream of an ATG start codon that ensures efficient tranlation initiation. A SalI site (GTCGAC) immediately upstream of the ATG start codon (GTCGACATG) is an effective kozak sequence minicircle: Covalently closed circular plasmid derivatives in which the bacterial region has been removed from the parent plasmid by in vivo or in vitro site specific recombination or in vitro restriction digestion/ligation. Minicircle vectors are replication incompetent in bacterial cells mSEAP: Murine secreted alkaline phosphatase Nanoplasmid vector: Vector combining an RNA selectable marker with a R6K or ColE2 related replication origin. For example, NTC9385C, NTC9685C, NTC9385R, NTC9685R vectors and modifications disclosed in provisional patent application Ser. No. 61/743,219 entitled ‘DNA plasmids with improved expression’ (Docket DPWIE082912) and included herein by reference and the NTC9385C2 NTC9385C2a, NTC9385R2, NTC9385R2a and NTC9385R2b series intronic replicative minicircle vectors of the invention disclosed herein NTC7382 promoter: A chimeric promoter comprising the CMV enhancer-CMV promoter-HTLV R-synthetic rabbit β globin 3′ intron acceptor -exon 2-SRF protein binding site-kozak sequence, with or without an upstream SV40 enhancer. The creation and application of this chimeric promoter is disclosed in Williams JA Supra, 2008 NTC8385: NTC8385, NTC8485 and NTC8685 plasmids are antibiotic-free vectors that contain a short RNA (RNA-OUT) selection marker in place of the antibiotic resistance marker (kanR). The creation and application of these RNA-OUT based antibiotic-free vectors are disclosed in Williams, J A Supra, 2008 and included herein by reference NTC8485: NTC8485 is an antibiotic-free vector that contains a short RNA (RNA-OUT) selection marker in place of the antibiotic resistance marker (kanR). The creation and application of NTC8485 is disclosed in Williams, J A 2010 U.S. patent application Ser. No. 2010/0184158 and included herein by reference NTC8685: NTC8685 is an antibiotic-free vector that contains a short RNA (RNA-OUT) selection marker in place of the antibiotic resistance marker (kanR). The creation and application of NTC8685 is disclosed in Williams, J A Supra, 2010 and included herein by reference OD₆₀₀: optical density at 600 nm PAS: Primosomal assembly site. Priming of DNA synthesis on a single stranded DNA ssi site. ØX174 type PAS: DNA hairpin sequence that binds priA, which, in turn, recruits the remaining proteins to form the preprimosome [priB, dnaT, recruits dnaB (delivered by dnaC)], which then also recruits primase (dnaG), which then, finally, makes a short RNA substrate for DNA polymerase I. ABC type PAS: DNA hairpin binds dnaA, recruits dnaB (delivered by dnaC) which then also recruits primase (dnaG), which then, finally, makes a short RNA substrate for DNA polymerase I. See Masai et al, 1990 J Biol Chem 265:15134. For example, the R6K plasmid CpG free ssiA primosomal assembly site (SEQ ID NO:17) or alternative ØX174 type or ABC type primosomal assembly sites, such as those disclosed in Nomura et al Supra, 1991 PAS-BH: Primosomal assembly site on the heavy (leading) strand PAS-BH region: pBR322 origin region between ROP and PAS-BL (approximately pBR322 2067-2351) PAS-BL: Primosomal assembly site on the light (lagging) strand PBS: Phosphate buffered Saline PCR: Polymerase Chain Reaction pDNA: Plasmid DNA pINT pR pL vector: The pINT pR pL integration expression vector is disclosed in Luke et al 2011 Mol Biotechnol 47:43 and included herein by reference. The target gene to be expressed is cloned downstream of the pL1 promoter. The vector encodes the temperature inducible cI857 repressor, allowing heat inducible target gene expression P_(L) promoter: Lambda promoter left. P_(L) is a strong promoter that is repressed by the cI repressor binding to OL1, OL2 and OL3 repressor binding sites. The temperature sensitive cI857 repressor allows control of gene expression by heat induction since at 30° C. the cI857 repressor is functional and it represses gene expression, but at 37-42° C. the repressor is inactivated so expression of the gene ensues Plasmid: An extra chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently from the chromosomal DNA pMB1 RNAI: pMB1 plasmid origin encoded RNAI (SEQ ID NO: 33; SEQ ID NO: 34) that represses RNAII regulated targets such as those described in Grabherr R, Pfaffenzeller I. 2006 US patent application US20060063232 and Cranenburgh R M. 2009; U.S. Pat. No. 7,611,883 P_(min): Minimal 678 by pUC replication origin SEQ ID NO:39 and functional variants with base substitutions and/or base deletions. Vectors described herein incorporating P_(min) include NTC9385P2(0.85)-01, NTC9385P2(0.85)-02, NTC9385P2a(0.85)-01, NTC9385P2a(0.85)-02 Pol: Polymerase polyA: Polyadenylation signal or site. Polyadenylation is the addition of a poly(A) tail to an RNA molecule. The polyadenylation signal is the sequence motif recognized by the RNA cleavage complex. Most human polyadenylation sites contain an AAUAAA motif and conserved sequences 5′ and 3′ to it. Commonly utilized polyA sites are derived from the rabbit β globin (NTC8485; FIG. 1), bovine growth hormone (gWIZ; pVAX1), SV40 early, or SV40 late polyA signals pUC replication origin: pBR322-derived replication origin, with G to A transition that increases copy number at elevated temperature and deletion of the ROP negative regulator pUC plasmid: Plasmid containing the pUC origin R6K plasmid: NTC9385R, NTC9685R, NTC9385R2-O1, NTC9385R2-O2, NTC9385R2a-O1, NTC9385R2a-O2, NTC9385R2b-O1, and NTC9385R2b-O2vectors as well as modifications and alternative vectors containing a R6K replication origin that were disclosed in provisional patent application Ser. No. 61/743,219 entitled ‘DNA plasmids with improved expression’ (Docket DPWIE082912) and included herein by reference. Alternative R6K vectors known in the art including, but not limited to, pCOR vectors (Gencell), pCpGfree vectors (Invivogen), and CpG free University of Oxford vectors including pGM169 R6K replication origin: a region which is specifically recognized by the plasmid-specified Rep protein to initiate DNA replication. Includes but not limited to R6K replication origin sequence disclosed SEQ ID NO:11: R6K Origin, and CpG free versions (SEQ ID NO:12) as disclosed in Drocourt et al U.S. Pat. No. 7,244,609 and incorporated herein by reference R6K replication origin-RNA-OUT bacterial region: Contains a R6K replication origin for propagation and the RNA-OUT selection marker (e.g. SEQ ID NO:26; SEQ ID NO:27; SEQ ID NO:28) Rep: Replication Replicative minicircle: Covalently closed circular plasmid in which the replication origin and optionally the selection marker are encoded within an intron of a eukaryotic region. For dual eukaryotic region vectors, the replication origin is cloned within one or more introns within the vector. Replicative minicircle vectors are replication competent in bacterial cells but not eukaryotic cells. Replicative minicircle vectors may contain a spacer region between the eukaryotic region sequences that are typically separated by the replication origin and selectable marker in plasmid vectors. This spacer region preferably is 500 by or less and may encode bacterial or eukaryotic selectable markers, bacterial transcription terminators, eukaryotic transcription terminators, boundary elements, S/MARs, RNA Pol I or RNA Pol III expressed sequences or other functionalities Rep protein dependent plasmid: A plasmid in which replication is dependent on a replication (Rep) protein provided in Trans. For example, R6K replication origin, ColE2-P9 replication origin and Co1E2 related replication origin plasmids in which the Rep protein is expressed from the host strain genome. Numerous additional Rep protein dependent plasmids are known in the art, many of which are summarized in del Solar et al 1998 Microbiol. Mol. Biol. Rev 62:434-464 which is included herein by reference RNA-IN: Insertion sequence 10 (IS 10) encoded RNA-IN, an RNA complementary and antisense to RNA-OUT. When RNA-IN is cloned in the untranslated leader of a mRNA, annealing of RNA-1N to RNA-OUT reduces translation of the gene encoded downstream of RNA-IN RNA-IN regulated selection marker: A genomically expressed RNA-IN regulated selectable marker. In the presence of plasmid borne RNA-OUT (e.g. SEQ ID NO:21), expression of a protein encoded downstream of RNA-IN is repressed. An RNA-IN regulated selection marker is configured such that RNA-IN regulates either 1) a protein that is lethal or toxic to said cell per se or by generating a toxic substance (e.g. SacB), or 2) a repressor protein that is lethal or toxic to said bacterial cell by repressing the transcription of a gene that is essential for growth of said cell (e.g. murA essential gene regulated by RNA-IN tetR repressor gene). For example, genomically expressed RNA-IN-SacB cell lines for RNA-OUT plasmid propagation are disclosed in Williams, J A Supra, 2008 and included herein by reference. Alternative selection markers described in the art may be substituted for SacB RNA-OUT: Insertion sequence 10 (IS10) encoded RNA-OUT transcription unit (SEQ ID NO:20), an antisense RNA that hybridizes to, and reduces translation of, the transposon gene expressed downstream of RNA-IN. The sequence of the RNA-OUT RNA (SEQ ID NO:21) and complementary RNA-IN SacB genomically expressed RNA-IN-SacB cell lines can be modified to incorporate alternative functional RNA-IN/RNA-OUT binding pairs such as those disclosed in Mutalik et al. 2012 Nat Chem Biol 8:447, including, but not limited to, the RNA-OUT A08/RNA-IN S49 pair, the RNA-OUT A08/RNA-IN S08 pair, and CpG free modifications of RNA-OUT A08 that modify the CG in the RNA-OUT 5′ TTCGCT sequence to a non-CpG sequence RNA-OUT Selectable marker: An RNA-OUT selectable marker DNA fragment including E. coli transcription promoter and terminator sequences flanking an RNA-OUT RNA. An RNA-OUT selectable marker, utilizing the RNA-OUT promoter and terminator sequences, that is flanked by DraIII and KpnI restriction enzyme sites, and designer genomically expressed RNA-IN-SacB cell lines for RNA-OUT plasmid propagation, are disclosed in Williams, J A Supra, 2008 (SEQ ID NO:20) and included herein by reference. The RNA-OUT promoter and terminator sequences flanking the RNA-OUT RNA (SEQ ID NO:21) may be replaced with heterologous promoter and terminator sequences. For example, the RNA-OUT promoter may be substituted with a CpG free promoter known in the art, for example the I-EC2K promoter or the P5/6 5/6 or P5/6 6/6 promoters disclosed in Williams, J A Supra, 2008 and included herein by reference RNA selectable marker: An RNA selectable marker is a plasmid borne expressed non translated RNA that regulates a chromosomally expressed target gene to afford selection. This may be a plasmid borne nonsense suppressing tRNA that regulates a nonsense suppressible selectable chromosomal target as described by Crouzet J and Soubrier F 2005 U.S. Pat. No. 6,977,174 included herein by reference. This may also be a plasmid borne antisense repressor RNA, a non limiting list included herein by reference includes RNA-OUT that represses RNA-IN regulated targets, pMB1 plasmid origin encoded RNAI (SEQ ID NO: 33; SEQ ID NO: 34) that represses RNAII regulated targets (Grabherr and Pfaffenzeller Supra, 2006; Cranenburgh RM. Supra, 2009), IncB plasmid pMU720 origin encoded RNAI that represses RNA II regulated targets (SEQ ID NO: 35; SEQ ID NO: 36; SEQ ID NO: 37; Wilson et al Supra, 1997), ParB locus Sok of plasmid Rl that represses Hok regulated targets, Flm locus FlmB of F plasmid that represses flmA regulated targets (Morsey M A, 1999 U.S. Pat. No. 5,922,583). An RNA selectable marker may be another natural antisense repressor RNAs known in the art such as those described in Wagner E G H, Altuvia S, Romby P. 2002. Adv Genet 46:361 and Franch T, and Gerdes K. 2000. Current Opin Microbiol 3:159. An RNA selectable marker may also be an engineered repressor RNAs such as synthetic small RNAs expressed SgrS, MicC or MicF scaffolds as described in Na D, Yoo S M, Chung H, Park H, Park J H, Lee S Y. 2013. Nat Biotechnol 31:170 ROP: Repressor of primer sacB: Structural gene encoding Bacillus subtilis levansucrase. Expression of sacB in gram negative bacteria is toxic in the presence of sucrose SEAP: Secreted alkaline phosphatase shRNA: Short hairpin RNA S/MAR: Scaffold/matrix attached region. Eukaryotic sequences that mediate DNA attachment to the nuclear matrix SR: Spacer region. As used herein, spacer region is the region linking the 5′ and 3′ ends of the eukaryotic region sequences. The eukaryotic region 5′ and 3′ ends are typically separated by the replication origin and selectable marker. In simple single RNA Pol II transcription vectors this will be between the RNA Pol II promoter region (5′ to either a promoter, enhancer, boundary element, S/MAR) and the RNA Pol II polyA region (3′ to either a polyA sequence, eukaryotic terminator sequence, boundary element, S/MAR). For example, in NTC8485 (FIG. 1) the 1492 by spacer region is the region between NheI site at 3737 and KpnI site at 1492. In dual RNA Pol II transcription vectors, the eukaryotic sequences separated by the spacer will depend on the orientation of the two transcription elements. For example, with divergent or convergent RNA Pol II transcription units, the spacer region may separate two polyA sequences or two enhancers respectively. In RNA Pol II, RNA Pol III dual expression vectors, the spacer region may separate an RNA Pol II enhancer and a RNA Pol III promoter. In short spacer region replicative minicircle vectors of the invention, this spacer region preferably is 500 by or less and may encode bacterial or eukaryotic selectable markers, bacterial transcription terminators, eukaryotic transcription terminators, boundary elements, S/MARs, RNA Pol I or RNA Pol HI expressed sequences or other functionalities ssi: Single stranded initiation sequences SV40 enhancer: Region containing the 72 by and optionally the 21 by repeats target antigen: Immunogenic protein or peptide epitope, or combination of proteins and epitopes, against which an immune response can be mounted. Target antigens may by derived from a pathogen for infectious disease applications, or derived from a host organism for applications such as cancer, allergy, or autoimmune diseases. Target antigens are well defined in the art. Some examples are disclosed in Williams, Supra, 2008 and are included herein by reference TE buffer: A solution containing approximately 10mM Tris pH 8 and 1 mM EDTA Transcription terminator: Bacterial: A DNA sequence that marks the end of a gene or operon for transcription. This may be an intrinsic transcription terminator or a Rho-dependent transcriptional terminator. For an intrinsic terminator, such as the trpA terminator, a hairpin structure forms within the transcript that disrupts the mRNA-DNA-RNA polymerase ternary complex. Alternatively, Rho-dependent transcriptional terminators require Rho factor, an RNA helicase protein complex, to disrupt the nascent mRNA-DNA-RNA polymerase ternary complex. Eukaryotic: PolyA sites are not ‘terminators’, instead internal cleavage at PolyA sites leaves an uncapped 5′ end on the 3′ UTR RNA for nuclease digestion. Nuclease catches up to RNA Pol II and causes termination. Termination can be promoted within a short region of the poly A site by introduction of RNA Pol H pause sites (eukaryotic transcription terminator). Pausing of RNA Pol II allows the nuclease introduced into the 3′ UTR mRNA after PolyA cleavage to catch up to RNA Pol II at the pause site. A nonlimiting list of eukaryotic transcription terminators know in the art include the C2×4 terminator (Ashfield R, Patel A J, Bossone S A, Brown H, Campbell R D, Marcu K B, Proudfoot N J. 1994. EMBO J 13:5656) and the gastrin terminator (Sato K, Ito R, Baek K H, Agarwal K, 1986. Mol. Cell. Biol. 6:1032). Terminator element may stabilize mRNA by enhancing proper 3′-end processing of mRNA (Kim D, Kim J D, Baek K, Yoon Y, Yoon J. 2003. Biotechnol Prog 19:1620) Transgene: Target antigen or protein that is cloned into a vector ts: Temperature sensitive μg: Microgram

Microliter UTR: Untranslated region of a mRNA (5′ or 3′ to the coding region) VARNA: Adenoviral virus associated RNA, including VARNAI (VAI or VAII) and or VARNAII (VAII or VA2) from any Adenovirus serotype, for example, serotype 2, serotype 5 or hybrids thereof VARNAI: Adenoviral virus associated RNAI, also referred to as VAI, or VA1, from any Adenovirus serotype, for example, serotype 2, serotype 5 or hybrids thereof Vector: A gene delivery vehicle, including viral (e.g. alphavirus, poxvirus, lentivirus, retrovirus, adenovirus, adenovirus related virus, etc) and nonviral (e.g. plasmid, midge, transcriptionally active PCR fragment, minicircles, bacteriophage, etc) vectors. These are well known in the art and are included herein by reference Vector backbone: Eukaryotic region and bacterial region of a vector, without the transgene or target antigen coding region

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention relates generally to plasmid DNA vector methods and compositions that improve plasmid manufacture and expression. The invention can be practiced to improve expression and manufacturing of vectors such as eukaryotic expression plasmids useful for gene therapy, genetic immunization and or interferon therapy. Improved plasmid expression is defined herein as improved expression level and/or expression duration. It is to be understood that all references cited herein are incorporated by reference in their entirety.

According to one preferred embodiment, the present invention provides compositions of short spacer region covalently closed super-coiled plasmid DNA eukaryotic vectors with improved transgene expression and E. coli manufacture, which comprises modifying the plasmid DNA to replace the replication origin in the vector spacer region with an intronic replication origin selected from the group consisting of an ColE2-P9 replication origin, ColE2 related replication origin, R6K replication origin, pUC replication origin and P_(min) pUC replication origin; transforming the modified plasmid DNA as necessary into a Rep protein producing bacterial cell line rendered competent for transformation; and isolating the resultant transformed bacterial cells. The modified plasmid produced from these cells is a ‘replicative minicircle’ vector with improved manufacture and transgene expression.

In one preferred embodiment, the vector backbone spacer region encoded replication origin is replaced with an intronic R6K replication origin to improve plasmid expression and manufacture. In another preferred embodiment, the vector backbone spacer region encoded replication origin is replaced with an intronic pUC replication origin to improve plasmid expression and manufacture. In another preferred embodiment, the vector backbone spacer region encoded replication origin is replaced with an intronic P_(min) pUC replication origin to improve plasmid expression and manufacture. In yet another preferred embodiment, the vector backbone spacer region encoded replication origin is replaced with an intronic ColE2 replication origin to improve plasmid expression and manufacture. In yet another preferred embodiment, the vector backbone spacer region encoded replication origin is replaced with an intronic CpG free ColE2 replication origin to improve plasmid expression and manufacture. In yet another preferred embodiment, the vector backbone spacer region encoded replication origin is replaced with an intronic CpG free R6K replication origin to improve plasmid expression and manufacture.

In yet another preferred embodiment, the replicative minicircle vector directly links the eukaryotic region sequences that are typically separated by the replication origin and selectable marker. In yet another preferred embodiment, the replicative minicircle vector eukaryotic region polyadenylation signal sequence is covalently linked directly to the enhancer of eukaryotic region promoter. In yet another preferred embodiment, a spacer region is included between the eukaryotic region sequences that are typically separated by the replication origin and selectable marker. In yet another preferred embodiment the spacer region between the sequences that are typically separated by the replication origin and selectable marker is 1 to 500 bp. In yet another preferred embodiment the spacer region between the sequences that are typically separated by the replication origin and selectable marker encode bacterial or eukaryotic selectable markers, bacterial transcription terminators, eukaryotic transcription terminators, boundary elements, S/MARs, RNA Pol I or RNA Pol III expressed sequences or other functionalities.

The methods of plasmid modification of the present invention have been surprisingly found to improve plasmid expression and manufacture.

Plasmid encoded transgene expression is preferably improved by employing specific constructs or compositions incorporated in a vector. According to one preferred embodiment, the present invention provides a composition for construction of a vector, comprising a ColE2 origin with at least 90% sequence identity to the sequences set forth as SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and a plasmid DNA encoded eukaryotic region, wherein the ColE2 origin is operably linked within an intron of the plasmid DNA encoded eukaryotic region. This novel vector configuration enables high yield manufacture of short spacer region vectors. It has also been surprisingly found that this intronic ColE2 origin improves plasmid encoded transgene expression. According to another preferred embodiment, the eukaryotic region has at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 31.

According to another preferred embodiment, the present invention provides a composition for construction of a vector, comprising an intronic R6K origin with at least 90% sequence identity to the sequences set forth as SEQ ID NO: 11, SEQ ID NO: 12, and a plasmid DNA encoded eukaryotic region, wherein the R6K origin is operably linked to an intron within the plasmid DNA encoded eukaryotic region. This novel vector configuration enables high yield manufacture of short spacer region vectors. It has also been surprisingly found that this intronic R6K origin improves plasmid encoded transgene expression. According to another preferred embodiment, the eukaryotic region has at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 30, SEQ ID NO: 31.

According to another preferred embodiment, the present invention provides a composition for construction of a vector, comprising an intronic pUC origin and a plasmid DNA encoded eukaryotic region, wherein the pUC origin is operably linked to an intron within the plasmid DNA encoded eukaryotic region. This novel vector configuration enables high yield manufacture of short spacer region vectors. It has also been surprisingly found that this intronic pUC origin improves plasmid encoded transgene expression. According to another preferred embodiment, the eukaryotic region has at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 30, SEQ ID NO: 31.

According to another preferred embodiment, the present invention provides a composition for construction of a vector, comprising an intronic P_(min) pUC origin with at least 90% sequence identity to the sequence set forth as SEQ ID. NO: 38, and a plasmid DNA encoded eukaryotic region, wherein the P_(min) pUC origin is operably linked to an intron within the plasmid DNA encoded eukaryotic region. This novel vector configuration enables high yield manufacture of short spacer region vectors. It has also been surprisingly found that this intronic P_(mm) pUC origin improves plasmid encoded transgene expression. According to another preferred embodiment, the eukaryotic region has at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 30, SEQ ID NO: 31.

The methods of plasmid modification of the present invention have been surprisingly found to improve plasmid expression in the target organism. Increased expression vectors may find application to improve the magnitude of DNA vaccination mediated antigen reactive B or T cell responses for preventative or therapeutic vaccination, increase RNA and or protein transgene levels to improve gene replacement therapy or gene knockdown therapy, increase plasmid based expression levels of DNA vector expressed therapeutic antibodies that neutralize infectious diseases such as influenza, HIV, malaria, hepatitis C virus, tuberculosis, etc.

As used herein, the term “sequence identity” refers to the degree of identity between any given query sequence, e.g. SEQ ID NO: 2, and a subject sequence. A subject sequence may, for example, have at least 90 percent, at least 95 percent, or at least 99 percent sequence identity to a given query sequence. To determine percent sequence identity, a query sequence (e.g. a nucleic acid sequence) is aligned to one or more subject sequences using any suitable sequence alignment program that is well known in the art, for instance, the computer program ClustalW (version 1.83, default parameters), which allows alignments of nucleic acid sequences to be carried out across their entire length (global alignment). Chema et al., 2003 Nucleic Acids Res., 31:3497-500. In a preferred method, the sequence alignment program (e.g. ClustalW) calculates the best match between a query and one or more subject sequences, and aligns them so that identities, similarities, and differences can be determined. Gaps of one or more nucleotides can be inserted into a query sequence, a subject sequence, or both, to maximize sequence alignments. For fast pair-wise alignments of nucleic acid sequences, suitable default parameters can be selected that are appropriate for the particular alignment program. The output is a sequence alignment that reflects the relationship between sequences. To further determine percent identity of a subject nucleic acid sequence to a query sequence, the sequences are aligned using the alignment program, the number of identical matches in the alignment is divided by the length of the query sequence, and the result is multiplied by 100. It is noted that the percent identity value can be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.

Turning now to the drawings, FIG. 1. shows an annotated map of the antibiotic free NTC8485 pUC origin expression vector with the locations of the pUC origin, PAS-BH primosomal assembly site, SV40 enhancer, HpaI site within the intron and other key elements indicated. The replication origin (PAS-BH and pUC origin) is from by 32 to the DraIII (1345) site (1313 by total). The antibiotic free RNA-OUT selection marker is between the DraIII (1345) and KpnI (1492) sites (147 by total). The bacterial region (trpA terminator, replication and RNA-OUT selection) of this vector is 1492 by (spacer region). Below the map an annotated sequence of the vector encoded HTLV-IR-Rabbit β globin hybrid intron (SEQ IND NO:1) is shown. The HTLV-I R derived 5′ intronic splice donor region and the Rabbit 1 globin 3′ splice acceptor region functionalities are separated by a HpaI site (GTTAAC, bold uppercase). The 5′ HTLV-I R derived splice donor (AGgtaagt; first 2 AG bases are exon 1) and rabbit β globin intron 1 derived 3′ splice acceptor (cagG; last G is exon 2) sites are double underlined. The 3′ splice acceptor poly-pyrimidine tract (ctttttctttttct) is single underlined. This poly-pyrimidine tract sequence was altered from the native rabbit β globin intron 1 sequence by replacing the native uppercase G and A residues with t (ctGtttcAtttct) to increase the poly-pyrimindine tract consensus. The rabbit β globin 3′ acceptor branch site (tgctgac) is single underlined. This intron is 225 by and is present in the NTC8385, NTC8485, NTC8685, NTC9385C, NTC9685C, NTC9385R, and NTC9685R vectors.

FIG. 2 shows bioinformatics analysis of an intron containing the gWIZ vector bacterial region (GBR2) encoded kanR selection marker-pUC origin. In this vector the kanR gene is antisense to the CMV promoter; the opposite sense orientation would be unacceptable due to safety concerns regarding the risk of kanR protein expression in the target organism. The kanR gene contains multiple cryptic splice acceptor and splice donor sites and potential sense and antisense promoters (not shown) predicted to interfere with intron function. The location and orientation of an experimentally verified cryptic pUC origin promoter (Lemp N A, Kiraoka K, Kasahara N, Logg C R. 2012. Nucleic Acids Res 40:7280) is shown (cryptic promoter). Splice signals were detected using the NetGene2 (Brunak, S., Engelbrecht, J., and Knudsen, S. 1991 J Mol Biol 220, 49-65) and Splicepredictor (Brendel, V., Xing, L. & Zhu, W. 2004. Bioinformatics 20, 1157-1169) programs while promoters were identified using the Softberry (Mount Kisco, N.Y.) TSSG and FPROM programs.

FIG. 3 shows bioinformatics analysis of introns containing the NTC9385P2 and NTC9385P2a bacterial region (P2) encoded RNA-OUT selection marker-pUC origin in both orientations. A cryptic 209 by exon derived from the pUC origin identified in A549 cells transfected with NTC9385P2-O2 [and NTC9385P2(0.85)-O2] is indicated as well as the cryptic splice acceptor and cryptic splice donor used in this cryptic exon. The location and orientation of an experimentally verified cryptic pUC origin promoter (Lemp et al., Supra,. 2012) is shown (cryptic promoter). Splice signals and promoters were detected as described in FIG. 2. The location of the regions removed in the NTC9385P2(0.85)-O1, NTC9385P2a(0.85)-O1, NTC9385P2(0.85)-O2 and NTC9385P2a(0.85)-O2 vectors are indicated (0.85 region 1 and 0.85 region 2).

FIG. 4 shows an annotated map of the NTC9385P2a-O1-EGFP and NTC9385P2a-O2-EGFP intronic pUC origin-RNA-OUT replicative minicircle expression vectors with the locations and orientations of the intronic RNA-OUT selection marker, pUC Replication origin (pUC origin) trpA terminator (SEQ ID NO: 29) and other key elements indicated. These vectors contain a 1436 by intron.

FIG. 5 shows plasmid quality from intronic pUC origin-RNA-OUT expression vectors NTC9385P2a-O1-EGFP, NTC9385P2a-O2-EGFP, NTC9385P2-O1-EGFP and NTC9385P2-O2-EGFP vectors versus a comparator backbone spacer region encoded pUC origin-RNA-OUT expression vector (NTC8385-EGFP). The top gel is a SYBR Green I prestain, the bottom gel is after SYBR Green II poststaining for 2 hrs followed by further electrophoresis to allow detection of shadow band or replication intermediates. SYBR Green I and II were obtained from Invitrogen (Carlsbad, Calif., USA).

FIG. 6 depicts the NTC9385R2a-O1-EGFP and NTC9385R2a-O2-EGFP intronic R6K origin-RNA-OUT replicative minicircle expression vectors with the locations and orientations of the intronic RNA-OUT selection marker, R6K gamma Replication origin (R6K mini-origin) trpA terminator (SEQ ID NO: 27) and other key elements indicated. These vectors contain a 685 by intron.

FIG. 7 depicts the NTC9385C2a-O1-EGFP and NTC9385C2a-O2-EGFP intronic ColE2 origin-RNA-OUT replicative minicircle expression vectors with the locations and orientations of the intronic RNA-OUT selection marker, ColE2-P9 Replication origin (Replication origin) primosomal assembly site (SEQ ID NO: 24) and other key elements indicated. These vectors contain a 499 by intron.

FIG. 8 shows plasmid quality from Table 6 fermentations of intronic R6K origin-RNA-OUT expression vectors NTC9385R2-O1-EGFP, NTC9385R2-O2-EGFP, NTC9385R2a-O1-EGFP and NTC9385R2a-O2-EGFP vectors, versus a comparator backbone spacer region encoded R6K origin-RNA-OUT expression vector (NTC9385R-EGFP). The gel is a SYBR Green I prestain. No replication intermediates or shadow band were detected after SYBR Green H poststain for 2 hrs followed by further electrophoresis.

FIG. 9 depicts the NTC9385R2b-O2-EGFP intronic R6K origin- spacer region RNA-OUT replicative minicircle expression vectors with the locations and orientations of the spacer region RNA-OUT selection marker, intronic R6K gamma Replication origin (R6K mini-origin SEQ ID NO: 11) trpA terminator (SEQ ID NO: 27) and other key elements indicated. This vector contains a 539 by intron.

FIG. 10 shows the pMB 1 and ColE 1 RNA I antisense repressor RNA. The RNAI promoter (−35 and −10) and RNAI antisense repressor RNA (italics; SEQ ID NO:33) is shown as well the location of the pUC high copy number G to A mutation

FIG. 11 shows an IncB RNAI based RNA selection marker. A) Genomically expressed target of RNAI RNA selection marker (SEQ ID NO: 37). Plasmid expressed RNAI binding to the pseudoknot in the complementary genomically expressed RNAII target prevents translation of the downstream SacB gene, conferring sucrose resistance. The RNAI −10 and −35 promoter elements are mutated to prevent RNAI expression. B) Structure of plasmid expressed IncB RNAI RNA selection marker (SEQ ID NO: 36) encoding the IncB RNAI antisense repressor (SEQ ID NO: 35).

The invention also relates to compositions and methods for producing short spacer region replicative minicircle plasmids with dramatically improved manufacturing yields and simplified manufacturing compared to alternative short spacer region vectors such as minicircles. The present invention also provides sequences that, when introduced into a vector backbone, increase plasmid expression.

The surprising observation that a ColE2, R6K, pUC or P_(min) pUC replication origin can be inserted into an intron of a eukaryotic RNA Pol II transcription unit without decreasing intron efficiency or transgene expression is disclosed.

As described herein, plasmid expression is improved by replacement of the vector backbone spacer region encoded replication origin with an R6K origin in an intron of a eukaryotic RNA Pol II transcription unit. In yet another preferred embodiment, the R6K origin is CpG free. In yet another preferred embodiment, the R6K origin is included with an RNA-OUT selection marker.

In yet another preferred embodiment, plasmid expression is improved by replacement of the vector backbone spacer region encoded replication origin with a ColE2 origin in an intron of a eukaryotic RNA Pol II transcription unit. In yet another preferred embodiment, the ColE2 origin is CpG free. In yet another preferred embodiment, the ColE2 origin is included with an RNA-OUT selection marker. In yet another preferred embodiment, the Co1E2 origin is included with a primosome assembly site.

In yet another preferred embodiment, plasmid expression is improved by replacement of the vector backbone spacer region encoded replication origin with a pUC origin in an intron of a eukaryotic RNA Pol H transcription unit. In yet another preferred embodiment, the pUC origin is included with an RNA-OUT selection marker.

In yet another preferred embodiment, plasmid expression is improved by replacement of the vector backbone spacer region encoded replication origin with a P_(min) pUC origin in an intron of a eukaryotic RNA Pol H transcription unit. In yet another preferred embodiment, the P_(min) pUC origin is included with an RNA-OUT selection marker.

EXAMPLES

The methods of the invention are further illustrated by the following examples. These are provided by way of illustration and are not intended in any way to limit the scope of the invention.

Example 1: pUC, R6K and ColE2 replication origin plasmid replication and production pUC origin vector replication and production background: The vast majority of therapeutic plasmids use the pUC origin which is a high copy derivative of the pMB1 origin (closely related to the ColE I origin). For pMB I replication, plasmid DNA synthesis is unidirectional and does not require a plasmid borne initiator protein. The pUC origin is a copy up derivative of the pMB1 origin that deletes the accessory ROP (rom) protein and has an additional temperature sensitive mutation that destabilizes the RNAI/RNAII interaction. Shifting of a culture containing these origins from 30 to 42° C. leads to an increase in plasmid copy number. pUC plasmids can be produced in a multitude of E. coli cell lines. pUC plasmid propagation and fermentations reported herein were performed using cell line NTC48165 =DH5α dcm attX::P5/6 6/6-RNA-IN- SacB or NTC54208=XL1Blue dcm attA::P5/6 6/6-RNA-IN- SacB the creation of which are disclosed in Carnes A E, Luke J M, Vincent J M, Schukar A, Anderson S, Hodgson C P, and Williams J A. 2011 Biotechnol Bioeng 108:354-363 and included herein by reference.

R6K origin vector replication and production background: The R6K gamma plasmid replication origin requires a single plasmid replication protein n that binds as a monomer to multiple repeated ‘iteron’ sites (seven core repeats containing TGAGNG consensus) and as a dimer to repressive sites [TGAGNG (dimer repress) as well as to iterons with reduced affmity]. Various host factors are used including IHF, DnaA, and primosomal assembly proteins DnaB, DnaC, DnaG (Abhyankar et al, 2003 J Biol Chem 278:45476-45484). The R6K core origin contains binding sites for DnaA and IHF that affect plasmid replication (n, IHF and DnaA interact to initiate replication).

Different versions of the R6K gamma replication origin have been utilized in various eukaryotic expression vectors, for example pCOR vectors (Soubrier et al 1999, Gene Therapy 6:1482) and a CpG free version in pCpGfree vectors (Invivogen, San Diego, Calif.), and pGM169 (University of Oxford). Incorporation of the R6K replication origin per se does not improve expression levels compared to an optimized pUC origin vector (Soubrier et al Supra, 1999). However, use of a conditional replication origin such as R6K gamma that requires a specialized cell line for propagation adds a safety margin since the vector will not replicate if transferred to a patients endogenous flora.

A highly minimalized R6K gamma derived replication origin (SEQ ID NO:11) that contains core sequences required for replication (including the DnaA box and sub 1-3 sites; Wu et al, 1995. J Bacteriol. 177: 6338-6345), but with the upstream n dimer repressor binding sites and downstream n promoter deleted (by removing one copy of the iterons) was disclosed in provisional patent application Ser. No. 61/743,219 entitled ‘DNA plasmids with improved expression’ (Docket DPWIE082912) and included herein by reference. The NTC9385R vector backbone including this minimalized R6K origin and the RNA-OUT AF selection marker in the spacer region, was disclosed in provisional patent application Ser. No. 61/743,219 entitled ‘DNA plasmids with improved expression’ (Docket DPWIE082912) and included herein by reference.

Typical R6K production strains incorporate the n protein derivative PIR116 that contains a P106L substitution that increases copy number (by reducing 17 dimerization; n monomers activate while 17 dimers repress). Fermentation results with pCOR (Soubrier et al Supra, 1999) and pCpG plasmids (Hebel H L, Cai Y, Davies L A, Hyde S C, Pringle I A, Gill D R. 2008. Mol Ther 16: S110) were low, around 100 mg/L in PIR116 cell lines.

Mutagenesis of the pir-116 replication protein and selection for increased copy number has been used to make new production strains. For example, the TEX2pir42 strain contains a combination of P106L and P42L. The P42L mutation interferes with DNA looping replication repression. The TEX2pir42 cell line improved copy number and fermentation yields with pCOR plasmids with reported yields of 205 mg/L (Soubrier F. Circular DNA molecule having a conditional origin of replication, process for their preparation and their use in gene therapy. World Patent Application WO2004033664, 2004).

Other combinations of 17 copy number mutants have been shown to improve copy number. This includes ‘P42L and P113S’ and ‘P42L, P106L and F107S’ (Abhyankar et al 2004. J Biol Chem 279:6711-6719).

R6K plasmid propagation and fermentations reported herein were performed using heat inducible ‘P42L, P106L and F107S’ n copy number mutant cell line NTC711231 the creation of which is disclosed in provisional patent application Ser. No. 61/743,219 entitled ‘DNA plasmids with improved expression’ (Docket DPWIE082912) and included herein by reference.

ColE2 origin vector replication and production background: The ColE2 replication origin (for example, ColE2-P9) is highly conserved across the ColE2-related plasmid family. Fifteen members are compared in Hiraga et al Supra, 1994, and fifty three ColE2 related plasmid members including ColE3 are compared in Yagura et al Supra, both references are included herein by reference. Plasmids containing this origin are normally 10 copies/cell (low copy number). For application in gene therapy vectors, the copy number of ColE2 replication origin vectors needs to be improved dramatically.

Expression of the ColE2-P9 replication (Rep) protein is regulated by antisense RNA (RNAI). Copy number mutations have been identified that interfere with this regulation and raise the copy number to 40/cell (Takechi et al 1994 Mol Gen Genet 244:49-56).

ColE2 plasmid propagation and fermentations were performed using heat inducible ‘G194D’ Rep protein copy number mutant cell line NTC710351 the creation of which is disclosed in provisional patent application Ser. No. 61/743,219 entitled ‘DNA plasmids with improved expression’ (Docket DPWIE082912) and included herein by reference.

The following vectors including NTC9385C containing the minimal Co1E2-P9 origin (Yagura and Itoh 2006 Biochem Biophys Res Commun 345:872-877) and various origin region modifications were disclosed in provisional patent application Ser. No. 61/743,219 entitled ‘DNA plasmids with improved expression’ (Docket DPWIE082912) and included herein by reference.

+7-ssiA: This combines the ColE2 origin (+7) (SEQ ID NO:13) with ssiA from plasmid R6K (SEQ ID NO:17). Thus ssiA vectors contain, in addition to the ColE2-P9 origin, a downstream primosome assembly site. Like most plasmid origins, the ColE2 origin contains a primosomal assembly site about 100 by downstream of the origin (Nomura et al Supra, 1991). This site primes lagging strand DNA replication (Masai et al 1990 J Biol Chem 265:15124-15133) which may improve plasmid copy number or plasmid quality. The ColE2 PAS (ssiA) is similar to PAS-BH (ColE1 ssiA =PAS-BL Marians et al 1982 JBiol Chem 257:5656-5662) and both sites (and PAS-BH) are CpG rich 0X174 type PAS. A CpG free PAS (ssiA from R6K; Nomura et al Supra, 1991; SEQ ID NO:17) that acts as a dnaA, dnaB dnaC (ABC) primosome on a dnaA box hairpin sequence (Masai et al Supra, 1990) was selected for inclusion in the +7-ssiA vectors. Alternative ABC or 0X174 type PAS sequences are functionally equivalent to ssiA from R6K, and may be substituted for ssiA in these ColE2 replication origin vectors. +7 CpG free-ssiA (SEQ ID NO:18): This combines the ColE2 replication origin (+7 CpG free) (SEQ ID NO:16) with ssiA from plasmid R6K (SEQ ID NO:17). The single CpG in the ColE2 replication origin was removed from the vector by site directed mutagenesis.

Yagura et al Supra, 2006 have demonstrated that the Min ColE2 Replication origin (SEQ ID NO:14, which is reverse complement of residues 7-38, in FIG. 1 of Yagura et al Supra, 2006) can be further deleted without eliminating replication function. Yagura et al, Supra, 2006, demonstrated that the core sequence is residues 8-35, with residues 5-36 are required for full activity. The +7 ColE2 Replication origin (SEQ ID NO:13; which is the reverse complement of residues 0-44 in FIG. 1 of Yagura et al Supra, 2006) could therefore be reduced to span residues 8-35 or 5-36 of FIG. 1 of Yagura et al Supra, 2006 (SEQ ID NO:15). Such vectors should replicate similarly to the disclosed vectors. As well, a number of base changes can be made within the core ColE2 origin 8-34 region that do not affect ColE2 replication (see changes to residues that retain function in Table 2; Yagura et al Supra, 2006).

The +7(CpG free)-ssiA ColE2 origin (SEQ ID NO 18) or +7(CpG free) ColE2 origin (SEQ ID NO 16) are smaller CpG free replication origin alternatives to the 260 by CpG free R6K replication origins (SEQ ID NO:12). CpG free ColE2 origins may be utilized to construct CpG free plasmid vectors. Combinations of a CpG free ColE2 or R6K replication origin with a CpG free RNA-OUT selection marker (SEQ ID NO: 22) may be utilized to construct antibiotic free CpG free bacterial regions for CpG free plasmid vectors (e.g. SEQ ID NO:25; SEQ ID NO:28).

Use of a conditional replication origin such as these ColE2 origins that requires a specialized cell line for propagation adds a safety margin since the vector will not replicate if transferred to a patients endogenous flora.

Example 2: NTC9385P2, NTC9385P2a, NTC9385C2, NTC9385C2a, NTC9385R2, and NTC9385R2a vector construction

A series of AF eukaryotic expression vectors incorporating intronic AF- pUC origin, AF- R6K origin or AF-ColE2 replication origins are disclosed.

FIG. 2 shows bioinformatics analysis of an intron containing the gWIZ vector bacterial region (GBR2) encoded kanR selection marker-pUC origin. This intron is predicted have reduced splicing efficiency and splicing precision due to the.presence of numerous splice acceptor sites, splice donor sites, and eukaryotic promoters in the kanR gene. Replacement of the kanR gene with the RNA-OUT antibiotic free marker results in an improved intron (FIG. 3) since the RNA-OUT sequence is not predicted to contain splice acceptor sites, splice donor sites, or eukaryotic promoters in either orientation.

However, the pUC origin does contain an experimentally verified cryptic eukaryotic promoter (FIG. 3) which likely would interfere with intron function. In addition, the close proximity of the pUC origin to the CMV enhancer repeats in an intronic vector is predicted to result in aberrant replication termination, resulting in replication intermediates which unacceptably reduce plasmid quality (Levy J. 2004. US Patent 6709844). So an intronically located pUC origin would be expected to interfere with eukaryotic intron function, and plasmid production quality.

The R6K and ColE2 origins do not contain predicted splice acceptor sites, splice donor sites, or eukaryotic promoters in either orientation. Replacement of the pUC origin with the R6K or ColE2 origins results in a improved intron design since the RNA-OUT-R6K and RNA-OUT-ColE2 bacterial region is not predicted to contain splice acceptor sites, splice donor sites, or eukaryotic promoters in either orientation.

NTC9385P2 and NTC9385P2a pUC origin replicative minicircle vectors: NTC8485-EGFP (FIG. 1) disclosed in Williams, JA Supra, 2010 contains the CMV enhancer and promoter upstream of a chimeric HTLV-IR rabbit β globin intron (SEQ ID NO: 1). The NTC8485-EGFP vector (FIG. 1) was linearized with HpaI which cuts internally within the intron (FIG. 1; SEQ ID NO: 1) leaving a blunt end. The pUC origin-RNA-OUT bacterial region (SEQ ID NO: 29) was excised from NTC8385 by digestion with Nhel (4 by protruding 5′ sticky end was blunted by end filling using klenow enzyme) and KpnI (4 by recessed 5′ sticky end was blunted by end chewing using T4 DNA polymerase enzyme). The two fragments were ligated and clones in either orientation and recombinant clones (NTC8485P2-O1-EGFP or NTC8485P2-02-EGFP) identified by restriction mapping and confirmed by DNA sequencing.

The NTC8485 encoded bacterial region and CMV enhancer encoded boundary element (NheI site to Spel site; FIG. 1) was removed by digestion of NTC8485P2-O1-EGFP and NTC8485P2-O2-EGFP with NheI and XbaI and subsequent ligation (NheI and XbaI have compatible 4 by sticky ends). Recombinant clones (NTC9385P2-O1-EGFP or NTC9385P2-O2-EGFP respectively) were identified by restriction mapping and confirmed by DNA sequencing.

The NTC8485 encoded bacterial region (NheI site to XbaI site; FIG. 1) was removed by digestion of NTC8485P2-O1-EGFP and NTC8485P2-O2-EGFP with NheI and XbaI and subsequent ligation (NheI and XbaI have compatible 4 by sticky ends). Recombinant clones (NTC9385P2a-O1-EGFP or NTC9385P2a-O2-EGFP respectively; FIG. 4) were identified by restriction mapping and confirmed by DNA sequencing.

The construction and isolation of these four NTC9385P clones demonstrates that the pUC origin and RNA-OUT selection marker can both function when located in an intron, in either orientation. Plasmid quality was evaluated by agarose gel analysis of plasmid preps from the four intronic pUC origin-RNA-OUT vectors, and the parent backbone spacer region encoded pUC-RNA-OUT vector NTC8385. Surprisingly, plasmid quality was high, and no replication intermediates were identified (FIG. 5) despite the close proximity of the pUC origin to the CMV enhancer (Levy J Supra, 2004).

NTC9385R2 and NTC9385R2a clones: The NTC8485-EGFP vector (FIG. 1) was linearized with HpaI which cuts internally within the intron (FIG. 1; SEQ ID NO: 1) leaving a blunt end. The R6K origin-RNA-OUT bacterial region (SEQ ID NO: 27) was excised from NTC9385R by digestion with Nhel (4 by protruding 5′ sticky end was blunted by end filling using klenow enzyme) and KpnI (4 by recessed 5′ sticky end was blunted by end chewing using T4 DNA polymerase enzyme). The two fragments were ligated and clones in either orientation (NTC8485R2-O1-EGFP or NTC8485R2-O2-EGFP) identified by restriction mapping and confirmed by DNA sequencing.

The NTC8485 encoded bacterial region and CMV enhancer encoded boundary element (NheI site to Spel site; FIG. 1) was removed by digestion of NTC8485R2-O1-EGFP and NTC8485R2-O2-EGFP with NheI and XbaI and subsequent ligation (NheI and XbaI have compatible 4 by sticky ends). Recombinant clones (NTC9385R2-O1-EGFP or NTC9385R2-O2-EGFP respectively) were identified by restriction mapping and confirmed by DNA sequencing.

The NTC8485 encoded bacterial region (NheI site to XbaI site; FIG. 1) was removed by digestion of NTC8485R2-O1-EGFP and NTC8485R2-O2-EGFP with NheI and XbaI and subsequent ligation (NheI and XbaI have compatible 4 by sticky ends). Recombinant clones (NTC9385R2a-O1-EGFP or NTC9385R2a-O2-EGFP respectively; FIG. 6) were identified by restriction mapping and confirmed by DNA sequencing.

The construction and isolation of these four NTC9385R clones demonstrates that the R6K origin and RNA-OUT selection marker can both function when located in an intron, in either orientation. Plasmid quality was evaluated by agarose gel analysis of plasmid preps from the four intronic R6K origin-RNA-OUT vectors. Surprisingly, plasmid quality was high, and no replication intermediates were identified (not shown).

NTC9385C2 and NTC9385C2a clones: The NTC8485-EGFP vector (FIG. 1) was linearized with HpaI which cuts internally within the intron (FIG. 1; SEQ ID NO: 1) leaving a blunt end. The ColE2 origin-RNA-OUT bacterial region (SEQ ID NO: 24) was excised from NTC9385C by digestion with NheI (4 by protruding 5′ sticky end was blunted by end filling using klenow enzyme) and KpnI (4 by recessed 5′ sticky end was blunted by end chewing using T4 DNA polymerase enzyme). The two fragments were ligated and clones in either orientation and recombinant clones (NTC8485C2-O1-EGFP or NTC8485C2-O2-EGFP) identified by restriction mapping and confirmed by DNA sequencing.

The NTC8485 encoded bacterial region and CMV enhancer encoded boundary element (NheI site to Spel site; FIG. 1) was removed by digestion of NTC8485C2-O1-EGFP and NTC8485C2-O2-EGFP with NheI and XbaI and subsequent ligation (NheI and XbaI have compatible 4 by sticky ends). Recombinant clones (NTC9385C2-O1-EGFP or NTC9385C2-O2-EGFP respectively) were identified by restriction mapping and confirmed by DNA sequencing.

The NTC8485 encoded bacterial region (NheI site to XbaI site; FIG. 1) was removed by digestion of NTC8485C2-O1-EGFP and NTC8485C2-O2-EGFP with NheI and XbaI and subsequent ligation (NheI and XbaI have compatible 4 by sticky ends). Recombinant clones (NTC9385C2a-O1-EGFP or NTC9385C2a-O2-EGFP respectively; FIG. 7) were identified by restriction mapping and confirmed by DNA sequencing.

The construction and isolation of these four NTC9385C clones demonstrates that the Co1E2 origin and RNA-OUT selection marker can both function when located in an intron, in either orientation. Plasmid quality was evaluated by agarose gel analysis of plasmid preps from the four intronic Co1E2 origin-RNA-OUT vectors. Surprisingly, plasmid quality was high, and no replication intermediates were identified (not shown).

Summary: The NTC9385P2, NTC9385P2a, NTC9385C2, NTC9385C2a, NTC9385R2, and NTC9385R2a replicative minicircle vectors are just a few possible nonlimiting intronic bacterial region replicative minicircle vector configurations. Many alternative vector configurations incorporating the novel intronic pUC, R6K or ColE2 origin vector modifications may also be made, including but not limited to vectors with alternative selection markers, alternative promoters, alternative introns, alternative polyadenylation sequences, a spacer region preferably 500 by or less between the eukaryotic polyadenylation site and the eukaryotic promoter, a eukaryotic transcription terminator between the eukaryotic polyadenylation site and the eukaryotic promoter, S/MAR, boundary elements, multiple transcription units separated by a spacer region, and different orientations of the various vector-encoded elements or alternative R6K or ColE2 origins as described in Example 1.

An example strategy for cloning into the NTC9385P2, NTC9385P2a, NTC9385C2, NTC9385C2a, NTC9385R2, and NTC9385R2a vectors is outlined below.

GTCGAC ATG--------Gene of interest---- SalI                                  Stop codon------AGATCT                BglII

The ATG start codon (double underlined) may be immediately preceded by a unique Sall site (GTCGACATG). This SalI-ATG site is an effective kozak sequence for translational initiation. Alternatively, a kozak sequence-ATG (e.g. gccRccATG) may be included downstream of the Sall site. Alternatively, the SalI site may be downstream in frame with an optimized secretion sequence such as TPA.

For precise cloning, genes are copied by PCR amplification from clones, cDNA, or genomic DNA using primers with Sall (5′ end) and BglII (3′ end) sites. Alternatively, genes are synthesized chemically to be compatible with the unique Sall / BgllI cloning sites in these vectors.

For all vectors one or two stop codons (preferably TAA or TGA) may be included after the open reading frame, prior to the Bell site.

Example 3: NTC9385P2, NTC9385P2a, NTC9385C2, NTC9385C2a, NTC9385R2, and NTC9385R2a vector expression

To determine intronic replicative minicircle vector eukaryotic region function, expression levels were determined in vitro using the vector encoded EGFP transgene. EGFP mRNA, EGFP protein (EGFP fluorescence) and splice junctions were determined after plasmid transfection.

Adherent HEK293 (human embryonic kidney) and A549 (human lung carcinoma),cell lines were obtained from the American Type Culture Collection (Manassas, Va., USA). Cell lines were propagated in Dulbecco's modified Eagle's medium/F12 containing 10% fetal bovine serum and split (0.25% trypsin-EDTA) using Invitrogen (Carlsbad, Calif., USA) reagents and conventional methodologies. For transfections, cells were plated on 24-well tissue culture dishes. plasmids were transfected into cell lines using Lipofectamine 2000 following the manufacturer's instructions (Invitrogen, Carlsbad Calif.).

Total cellular lysates for EGFP determination were prepared by resuspending cells in cell lysis buffer (BD Biosciences Pharmingen, San Diego, CA, USA), lysing cells by incubating for 30 min at 37° C., followed by a freeze-thaw cycle at -80 ° C. Lysed cell supernatants were assayed for EGFP by FLX800 microplate fluorescence reader (Bio-Tek, Winooski, Vt., USA).

Cytoplasmic RNA was isolated from transfected HEK293 and A549 cells using the protein and RNA isolation system (PARIS kit, Ambion, Austin, Tex.) and quantified by A₂₆₀. Samples were DNase treated (DNA-free DNase; Ambion, Austin, Tex.) prior to reverse transcription using the Agpath-ID One step RT-PCR kit (Ambion, Austin, Tex.) with the EGFP transgene specific complementary strand primer EGFPR (FIG. 1). Intron splicing was determined by PCR amplification of the reverse transcribed cytoplasmic RNA with the EGFP5Rseq and CMVF5seq primers (FIG. 1). EGFP mRNA levels in the reverse transcribed cytoplasmic RNA were quantified by quantitative PCR using a TaqMan EGFP transgene 6FAM-probe-MGBNFQ probe and flanking primers EGFPR and EGFPF (FIG. 1) in a TaqMan Gene expression assay using Applied Biosystems (Foster City, Calif.) TaqMan reagents and the Step One Real Time PCR System. Methods and primer and probe sequences are described in Luke J M, Vincent J M, Du S X, Gerdemann U, Leen A M, Whalen R G, Hodgson C P, and Williams J A. 2011. Gene Therapy 18:334-343 included herein by reference. Linearized vector was used for the RT-PCR standard curve.

The results are summarized in Tables 1-5. In Table 1 EGFP expression in HEK293 and A549 cell lines after transfection with NTC8485-EGFP (backbone spacer region AF-pUC origin) or NTC8485 derivatives further including intronic AF-pUC, AF-ColE2 or AF-R6K origins (also with backbone spacer region AF-pUC origin) is shown. The ColE2 and R6K intronic bacterial regions had similar expression levels comparable to the unaltered intron in NTC8485, while expression from the intronic pUC origin was slightly reduced.

TABLE 1 Intron encoded RNA-OUT selection/replication origin does not prevent transgene expression NTC8485 NTC8485 A549 FU HEK293 FU Vector (all Construct vector spacer Vector Intron^(a) (T = 48 (T = 48 EGFP) ID # region^(a) (intron size) mean ± SD)^(b) mean ± SD)^(b) NTC8485 NTC-0200620 P-AF-SV40-BE HR -β 5886 ± 249 32628 ± 1015 (225bp intron) (1x)   (1x)   NTC8485C2- 073-030-1H P-AF-SV40-BE HR ←C AF→β 3638 ± 351 25231 ± 2124 O1 (499 bp intron) (0.62x) (0.77x) NTC8485C2- 073-030-1A P-AF-SV40-BE HR ←AF C →β 4144 ± 275 26233 ± 1842 O2 (499 bp intron) (0.70x) (0.80x) NTC8485R2- 073-036-1B P-AF-SV40-BE HR←T-R AF→β 3656 ± 240 23905 ± 679  O1 (685 bp intron) (0.62x) (0.73x) NTC8485R2- 073-036-1A P-AF-SV40-BE HR←AF R-T→β 4062 ± 249 22165 ± 1281 O2 (685 bp intron) (0.69x) (0.68x) NTC8485P2- 073-041-2L P-AF-SV40-BE HR← T-P-AF→β 2565 ± 294 20757 ± 1457 O1 (1436 bp intron) (0.44x) (0.64x) NTC8485P2- 073-041-2E P-AF-SV40-BE HR←AF P -T →β 2411 ± 320 15333 ± 1145 O2 (1436 bp intron) (0.41x) (0.47x) ^(a)trpA term = T; HTLV-IR = HR; B globin 3′ acceptor site = β; RNA-OUT sucrose selection marker = AF; pUC origin = P; R6K origin = R; ColE2 origin = C; BE = CMV boundary element (XbaI-SpeI fragment); SV40 = SV40 enhancer; ^(b)FU = Fluorescence units ( ) Mean FU standardized to NTC8485

Conversion of the NTC8485C2, NTC8485R2 and NTC8485P2 (pUC origin-AF backbone spacer region) vectors into replicative minicircles by removal of the pUC origin-AF backbone spacer region to create the corresponding NTC9385C2, NTC9385R2 and NTC9385P2 vectors (Example 2) dramatically increased expression compared to the NTC8485C2, NTC8485R2 and NTC8485P2 parent vectors (Table 2). This demonstrates that the replicative minicircles of the invention improve expression through removal of vector backbone spacer region encoded bacterial region.

TABLE 2 Improved expression with intron encoded RNA-OUT selection/replication origin Vector A549 FU ^(b) HEK293 FU ^(b) Vector (all Construct Spacer (T = 48 (T = 48 EGFP) ID # Region ^(a) Vector Intron ^(a) mean ± SD) mean ± SD) NTC8485 NTC- P-AF-SV40- HR -β 5886 ± 249 32628 ± 1015 0200620 BE (1x)   (1x)   NTC9385C 071-020-2D C-AF HR -β 8591 ± 168 35293 ± 1798 (1.46x) (1.08x) NTC8485C2- 073-030-1H P-AF-SV40- HR ←C AF→β 3638 ± 351 25231 ± 2124 O1 BE (0.62x) (0.77x) NTC8485C2- 073-030-1A P-AF-SV40- HR ←AF C →β 4144 ± 275 26233 ± 1842 O2 BE (0.70x) (0.80x) NTC9385C2- 073-032-5A None HR ← C AF→ β 6793 ± 521 24762 ± 1498 O1 (1.15x) (0.76x) NTC9385C2- 073-032-6A None HR ←AF C →β 7330 ± 692  24811± 1256 O2 (1.25x) (0.76x) NTC9385C2a- 073-032-7A None (BE) HR ← C AF→β 7515 ± 282 29444 ± 2193 O1 (1.28x) (0.90x) NTC9385C2a- 073-032-8A None (BE) HR ←AF C →β 7255 ± 322 27055 ± 1850 02 (1.23x) (0.83x) NTC9385R 071-025-2C R-AF HR -β 5813 ± 949 29822 ± 661  (0.99x) (0.91x) NTC8485R2- 073-036-1B P-AF-SV40- HR←T-R AF→β 3656 ± 240 23905 ± 679  O1 BE (0.62x) (0.73x) NTC8485R2- 073-036-1A P-AF-SV40- HR←AF R-T→β 4062 ± 249 22165 ± 1281 O2 BE (0.69x) (0.68x) NTC9385R2- 073-038-1A None HR← T-R-AF→β 10959 ± 1278 34521 ± 3694 O1 (1.86x) (1.06x) NTC9385R2- 073-038-2A None HR←AF R-T →β 10652 ± 567  31586 ± 1121 O2 (1.81x) (0.97x) NTC9385R2a- 073-038-3A None (BE) HR← T-R-AF→β 10699 ± 674  37603 ± 2671 O1 (1.82x) (1.15x) NTC9385R2a- 073-038-4A None (BE) HR←AF R-T →β 10251 ± 1343 34086 ± 1518 O2 (1.74x) (1.04x) NTC8485P2- 073-041-2L P-AF-SV40- HR← T-P-AF→β 2565 ± 294 20757 ± 1457 O1 BE (0.44x) (0.64x) NTC8485P2- 073-041-2E P-AF-SV40- HR←AF P -T →β 2411 ± 320 15333 ± 1145 O2 BE (0.41x) (0.47x) NTC9385P2- 073-043-1A None HR←AF P-T →β 5561 ± 497 21838 ± 589  O2 (0.94x) (0.67x) NTC9385P2a- 073-043-2A None (BE) HR←AF P -T →β 6291 ± 544 23808 ± 2411 O2 (1.07x) (0.73x) ^(a) trpA term = T; HTLV-IR = HR; B globin 3′ acceptor site = β; RNA-OUT sucrose selection marker = AF; pUC origin = P; R6K origin = R; ColE2 origin = C; BE = CMV boundary element (XbaI-SpeI fragment); SV40 = SV40 enhancer; ^(b) FU = Fluorescence units ( ) Mean FU standardized to NTC8485

Table 3 demonstrates that mRNA splicing is accurate and spliced mRNA export efficient with the intronic bacterial regions encoded in NTC9385C2, NTC9385R2 and NTC9385P2. A minor amount of a cryptic 209 by pUC origin derived exon was identified with NTC9385P2-02 (but not NTC9385P2-01) in A549 cells but not HEK293 cells (Table 3; 490 by band). The cryptic exon sequence was determined by sequencing of the PCR product and the cryptic 209 by exon utilized cryptic splice donor and acceptor sites within the pUC origin (FIG. 3).

TABLE 3 Intron functional analysis - Splicing accuracy and export efficiency EGFP % Predicted Actual RT-PCR EGFP spliced exon spliced exon # Plasmid Cell line (pg) mRNA^(c) size (unspliced) size (PCR) 1 NTC8685 HEK293 448.3 ± 38.7 0.74% 279 (514) 279 ^(a) 2 NTC9385C2-O1 HEK293  274.6 ± 10.3* 0.44% 279 (788) 279 ^(a) 3 NTC9385C2-O2 HEK293 227.4 ± 4.9* 0.41% 279 (788) 279 ^(a) 4 NTC9385R2-O1 HEK293  398.9 ± 14.8* 0.64% 279 (974) 279 ^(a) 5 NTC9385R2-O2 HEK293 350.7 ± 5.2* 0.57% 279 (974) 279 ^(a) 6 NTC9385P2-O2 HEK293 181.6 ± 5.5  0.30% 279 (974) 279 ^(a) 7 NTC8485P2-O1 HEK293 160.1 ± 12.3 0.27%  279 (1715) 279 ^(a) 8 NTC8685 A549 50.6 ± 4.3 0.128% 279 (514) 279 ^(a) 9 NTC9385C2-O1 A549 29.2 ± 1.2 0.082% 279 (788) 279   10 NTC9385C2-O2 A549 23.9 ± 1.4 0.074% 279 (788) 279   11 NTC9385R2-O1 A549 41.9 ± 1.6 0.116% 279 (974) 279   12 NTC9385R2-O2 A549 35.8 ± 2.2 0.096% 279 (974) 279 ^(a) 13 NTC9385P2-O2 A549 17.4 ± 0.6 0.050%  279 (1715) 279 ^(b) 14 NTC8485P2-O1 A549  7.0 ± 0.2 0.024%  279 (1715) 279   ^(a) Correct splice junction verified by DNA sequencing of PCR product ^(b) Faint extra bands at 490 and 650 bp, not present in 6 (HEK293 equivalent) or other samples ^(c)% of total cytoplasmic RNA that is EGFP mRNA

Table 4 further demonstrates robust expression is observed with all NTC9385C2, NTC9385R2 and NTC9385P2 replicative minicircle vectors (both orientations, with and without CMV boundary region). Overall, the highest expression is obtained with the R6K replicative minicircle vectors (NTC9385R2-O1; NTC9385R2-02; NTC9385R2a-O1; NTC9385R2a-O2).

TABLE 4 Intron vector expression efficiency A549 HEK293 Vector (T = 48 (T = 48 Vector (all Construct Spacer mean ± mean ± # EGFP) ID # Region^(a) Vector Intron^(a) SD) SD) 1 NTC9385C2- 073-032-5A None HR ←C AF→β  7581 ± 1145 20868 ± 9153 O1 2 NTC9385C2- 073-032-6A None HR ←AF C →β 6012 ± 503 12902 ± 2356 O2 3 NTC9385C2a- 073-032-7A BE HR ←C AF→β 6018 ± 979 13564 ± 799  O1 4 NTC9385C2a- 073-032-8A BE HR ←AF C →β 6633 ± 136 16119 ± 729  O2 5 NTC9385R2- 073-038-1A None HR←T-R AF→β 9626 ± 304 18627 ± 999  O1 6 NTC9385R2- 073-038-2A None HR←AF R-T→β 8513 ± 235 12660 ± 348  O2 7 NTC9385R2a- 073-038-3A BE HR←T-R AF→β 8295 ± 188 15601 ± 2550 O1 8 NTC9385R2a- 073-038-4A BE HR←AF R-T→β 8724 ± 188 19219 ± 1763 O2 9 NTC9385P2- 073-126-1A None HR← T-P-AF→β 6086 ± 704 16967 ± 2237 O1 10 NTC9385P2- 073-043-1A None HR←AF P-T →β 4941 ± 283 11604 ± 2580 O2 11 NTC9385P2a- 073-126-2A BE HR← T-P-AF→β 5277 ± 114 13073 ± 1779 O1 12 NTC9385P2a- 073-043-2A BE HR←AF P-T →β 5122 ± 608 11182 ± 870  O2 ^(a)trpA term = T; HTLV-IR = HR; B globin 3′ acceptor site = β; RNA-OUT sucrose selection marker = AF; pUC origin = P; R6K origin = R; ColE2 origin = C; BE = CMV boundary element (XbaI-SpeI fragment); SV40 = SV40 enhancer

Table 5 demonstrates in vivo expression after intradermal delivery with the intronic bacterial region vectors of the invention was improved compared to an optimized plasmid comparator (NTC8685). NTC9385R2a-O2 expression was improved 1.9-4.1 fold compared to NTC8685 while NTC9385P2a-O1 was improved 5-8 fold. NTC9385R2-O2 expression was also improved (1.3-1.5 fold compared to NTC8685) but less than NTC9385R2a-O2 suggesting that the CMV promoter derived boundary element adjacent to the spacer region is beneficial. Replicative minicircle expression is surprisingly much higher relative to plasmid comparator in vivo compared to in vitro (Table 5). While not limiting the application of the invention, this may be an unexpected benefit of removal of the large spacer region encoded replication origin and selection marker, perhaps through immediate spacer region directed heterochromatin formation that is more prevalent in vivo than in vitro. The improved expression level after intradermal delivery demonstrates the application of replicative minicircle vectors of the invention for cutaneous gene therapy applications, for example, for wound healing, burns, diabetic foot ulcer, or critical limb ischemia therapies using growth factors such as hypoxia inducible factor, hypoxia inducible factor 1 a, keratinocyte growth factor, vascular endothelial growth factor (VEGF), fibroblast growth factor-1 (FGF-1, or acidic FGF), FGF-2 (also known as basic FGF), FGF-4, placental growth factor (PIGF), angiotensin-1 (Ang-1), hepatic growth factor (HGF), Developmentally Regulated Endothelial Locus (Del-1), stromal cell derived factor-1 (SDF-1), etc.

TABLE 5 Replicative minicircle vector expression in vitro (lipofectamine) and in vivo (intradermal delivery with electroporation) HEK- ID + EP^(c) ID + EP^(c) ID + EP^(c) muSEAP SR A549 293 (pg/mL) (pg/mL) (pg/mL) Vector^(b) SR^(a) (bp) Intron^(a) (A₄₀₅)^(d) (A₄₀₅)^(d) T = 4 day T = 7 day T = 14 day NTC8685 T-VA1- 1695 HR-β 0.240 ± 3.002 ± 1.9 ± 1.2 6.7 ± 4.1& 5.0 ± 3.9 BH-P-AF 0.029 0.188 (1.0x) (1.0x) (1.0x) (1.0x) (1.0x) NTC9385P2a- None 0 HR T← P 0.467 ± 2.890 ± 9.5 ± 6.2 53.4 ± 34.8 ± O1 (BE) AF→-β 0.047 0.085 (5.0x) 51.5 29.6 (2.0x) (1.0x) (8.0 x) (7.0x) NTC9385R2a- None 0 HR←AF 0.409 ± 2.561 ± 6.5 ± 6.1 27.6 ± 9.5 ± 11.2 O2 (BE) R → T-β 0.039 0.038 (3.4x) 25.9 (1.9x) (1.7x) (0.9x) (4.1x) NTC9385R2- None 0 HR←AF 0.564 ± 2.999 ± 2.8 ± 3.8 8.8 ± 15.7 7.0 ± 8.5 O2 R → T-β 0.008 0.106 (1.5x) (1.3x) (1.4x) (2.4x) (1.0x) ^(a)Prokaryotic terminator = T; HTLV-IR = HR; B globin 3′ acceptor site = β; RNA-OUT = AF; pUC origin = P; R6Kγ origin = R; ColE2-P9 origin = C ^(b)P vectors produced in dcm-XL1Blue NTC54208; R vectors produced in dcm-R6K rep cell line NTC711231; C vectors produced in dcm-ColE2 rep cell line NTC710351 ^(c)Dose = 50 μg in 50 μL saline injected intradermally (ID) with EP. 6 mice/group. Mean ± SD pg/mL muSEAP on indicated day post EP reported. ( ) Mean muSEAP standardized to NTC8685 ^(d)muSEAP plasmid DNA transfected with Lipofectamine 2000. Mean ± SD 48 hr post transfection A₄₀₅ reported. ( ) Mean A₄₀₅ standardized to NTC8685

Reduction of the vector spacer region size as described herein by removal of the bacterial region replication origin and addition of an intronic R6K, ColE2, pUC or P_(min) pUC origin vectors of the invention will also increase the duration of in vivo expression since expression duration is improved with plasmid vectors in which the bacterial region is removed (minicircle) or replaced with a spacer region of up to at least 500 by (Lu et al. Supra, 2012). Thus the replicative minicircle vectors of the invention also have additional utility for applications requiring extended duration expression, such as: liver gene therapy using hydrodynamic delivery with transgenes such as a −1 antitrypsin (AAT) for AAT deficiency, Coagulation Factor VIII for Hemophilia A Therapy or Coagulation Factor IX for Hemophilia B Therapy etc: lung gene therapy with transgenes such as Cystic fibrosis transmembrane conductance regulator (CFTR) for cystic fibrosis etc; muscle gene therapy with transgenes such as the GNE gene for Hereditary inclusion body myopathies (HIBM), or dystrophin or dystrophin minigenes for duchenne muscular dystrophy (DMD).

The intronic replicative minicircles of the invention may optionally additionally encode a spacer region of at least 500 by between the eukaryotic region polyadenylation signal and the eukaryotic region promoter. This spacer region may include a number of functional sequences such as bacterial or eukaryotic selectable markers, bacterial transcription terminators, eukaryotic transcription terminators, boundary elements, S/MARs, RNA Pol I or RNA Pol III expressed sequences or other functionalities.

Example 4: Replicative minicircle vector fermentation production

Fermentation : Fermentations were performed using proprietary fed-batch media (NTC3019, HyperGRO media) in New Brunswick BioFlo 110 bioreactors as described (Carnes and Williams, Supra, 2011). The seed cultures were started from glycerol stocks or colonies and streaked onto LB medium agar plates containing 6% sucrose. The plates were grown at 30-32° C.; cells were resuspended in media, and used to provide approximately 0.1% inoculums for the fermentations that contained 0.5% sucrose to select for RNA-OUT plasmids.

Production hosts: Antibiotic-free pUC origin RNA-OUT plasmid fermentations were performed in E. coli strain XL1Blue [recAl endAl gyrA96 thi-1 hsdR17 supE44 relAl lac [F′proAB laclqZΔM15 Tn10 (Tet′)] (Stratagene, La Jolla, Calif.)] dcm or DH5a [F-Φ80lacZΔM15 Δ(lacZY A-argF) U169 recAl endAl hsdR17 (rK−, mK+) phoA supE44 λ-thi-1 gyrA96 relAl] (Invitrogen, Carlsbad, Calif.) dcm containing chromosomally integrated pCAH63-CAT RNA-IN-SacB (P5/6 6/6) as disclosed in Williams, J A Supra, 2008. SacB (Bacillus subtilis levansucrase) is a counterselectable marker which is lethal to E. coli cells in the presence of sucrose. Translation of SacB from the RNA-IN-SacB transcript is inhibited by plasmid encoded RNA-OUT. This facilitates plasmid selection in the presence of sucrose, by inhibition of SacB mediated lethality. These production strains are NTC54208=XL1Blue dcm attX::P5/6 6/6-RNA-IN- SacB and NTC48165=DH5a dcm attA::P5/6 6/6-RNA-IN-SacB

Antibiotic-free R6K plasmid propagation and fermentations were performed using pL promoter heat inducible ‘P42L, P106L and F107S’ n copy number mutant cell line NTC711231 the creation of which is disclosed in provisional patent application Ser. No. 61/743,219 entitled ‘DNA plasmids with improved expression’ (Docket DPWIE082912) and included herein by reference. NTC711231 is NTC54208-pR pL (OLl-G to T) P42L-P106L-F107S (P3-).

ColE2 plasmid propagation and fermentations were performed using pL promoter heat inducible ‘G194D’ Rep protein copy number mutant cell line NTC710351 the creation of which is disclosed in provisional patent application Ser. No. 61/743,219 entitled ‘DNA plasmids with improved expression’ (Docket DPWIE082912) and included herein by reference. NTC710351=NTC54208-pR pL (OL1-G to T) ColE2 rep G194D

Analytical Methods: Culture samples were taken at key points and at regular intervals during all fermentations. Samples were analyzed immediately for biomass (OD₆₀₀) and for plasmid yield. Plasmid yield was determined by quantification of plasmid obtained from Qiagen Spin Miniprep Kit preparations as described (Carnes and Williams, Supra, 2011). Briefly, cells were alkaline lysed, clarified, plasmid was column purified, and eluted prior to quantification. Agarose gel electrophoresis analysis (AGE) was performed on 0.8-1% Tris/acetate/EDTA (TAE) gels as described in Carnes and Williams, Supra, 2011.

Results: Fermentation yields are summarized in Table 6. The results demonstrated that the replicative minicircle vectors of the invention have efficient manufacture. Manufacture was effective with ColE2, R6K and pUC replicative minicircles with yield of 235-703 mg/L, up to >100 fold improved compared to reported yields of 5 mg/L with alternative short spacer region minicircle vectors (Kay et al, Supra, 2010). Additionally, the replicative minicircle vectors of the invention do not require the complicated difficult to scale expensive additional manufacturing steps required to remove the bacterial region between the eukaryotic polyA and promoter with minicircle vectors (Kay et al, Supra, 2010). This is because replicative minicircles have no bacterial region between the eukaryotic polyA and the promoter to remove, since replication and selection functions are now innocuously encoded in an intron within the eukaryotic transcription unit.

As well, fermentation plasmid DNA was high quality with ColE2, R6K and pUC replicative minicircles. A comparison of plasmid production with the 4 R6K intronic selection vectors (NTC9385R2-O1-EGFP; NTC9385R2-O2-EGFP; NTC9385R2a-O1-EGFP; NTC9385R2a-O2-EGFP) versus a standard R6K backbone spacer region vector NTC9385R-EGFP demonstrated no differences in yield (Table 6) or quality (FIG. 8).

TABLE 6 Intronic RNA-OUT AF selection plasmid fermentation yields^(a) Growth phase Production Specific phase final Production Plasmid yield Specific phase final Production size Vector (mg/L/ yield (mg/L/ biomass phase final Plsmid (kb) Origin type^(f) OD600) OD₆₀₀) (OD₆₀₀) yield (mg/L)^(e) NTC9385R- 2.4 R6K^(b) SR 1.4-1.5 4.4 89 392 EGFP NTC9385R2- 2.4 R6K^(b) Intron 1.4-1.5 6.3 66 414 O1-EGFP NTC9385R2- 2.4 R6K^(b) Intron 1.1-1.8 3.8 70 269 O2-EGFP NTC9385R2a- 2.5 R6K^(b) Intron 1.7-1.9 4.8 74 356 O1-EGFP NTC9385R2a- 2.5 R6K^(b) Intron 1.0-1.8 3.5 69 244 O2-EGFP NTC9385R2b- 2.4 R6K^(b) Intron 2.6-3.0 7.2 61 440 O2-EGFP (AF- SR) NTC9385C- 2.2 ColE2^(c) SR 0.6-1.0 5.8 115 672 EGFP NTC9385C2a- 2.3 ColE2^(c) Intron 0.8-1.1 3.7 87 323 O1-EGFP NTC9385C2a- 2.3 ColE2^(c) Intron 0.8-1.0 3.7 64 235 O2-EGFP NTC9385P2a- 3.2 pUC^(d) Intron 1.2-1.3 8.6 82 703 O1-EGFP NTC9385P2a(0. 2.9 pUC^(d) Intron 0.9-1.0 5.6 84 468 85)-O1-EGFP (0.85) NTC9385P2a(0. 2.9 pUC^(d) Intron  0.8-1.05 5.7 77 439 85)-O2-EGFP (0.85) NTC8385 2.8 pUC^(d) SR 0.9 6.4 105 667 (0.85)-EGFP (0.85) ^(a)30° C. growth phase to 50-60 OD600. Plasmid copy number then induced by temperature shift to 42° C. and subsequent 7-10.5 hr growth post induction (production phase) ^(b)R6K plasmids produced in cell line NTC711231 = NTC54208-pR pL (OL1-G to T) P42L-P106L-F107S (P3-). NTC54208 = XL1Blue dcm attλ::P5/6 6/6-RNA-IN-SacB ^(c)ColE2 plasmids produced in cell line NTC710351 = NTC54208-pR pL (OL1-G to T) ColE2 rep G194D ^(d)pUC plasmids produced in cell line NTC54208 = XL1Blue dcm attλ::P5/6 6/6-RNA-IN-SacB ^(e)a By comparison, minicircle manufacturing final volumetric yield are 5 mg/L (Kay et al, Supra, 2010) ^(f)SR = Replication origin and selection marker in spacer region. Intron = Replication origin and selection marker in intron. NTC9385R2b has RNA-OUT in SR and R6K origin in intron

Example 5: High level expression with replicative minicircle vectors modified to include and RNA selection marker or a eukaryotic transcriptional terminator in the spacer region

Minicircle vectors contain a spacer region between the eukaryotic region polyA and promoter sequences; this spacer region may be at least 500 by (Lu et al Supra, 2012). To determine if the spacer region may encode a selectable marker, bacterial transcription terminator, eukaryotic transcription terminator or other functionality, replicative minicircle vectors were created that included either a RNA-OUT selection marker or a Gastrin eukaryotic terminator. FIG. 9 shows NTC9385R2b-O2-EGFP, an example intronic R6K origin replicative minicircle vector in which the RNA-OUT RNA selection marker is located in the spacer region between the eukaryotic polyA and the eukaryotic promoter rather than within the intron. The vector has a 148 by spacer region, well below 500 bp. The vector was transfected into HEK293 and A549 cell lines, and EGFP expression and splicing were analyzed as described in Example 3. High level expression (Table 7) and accurate splicing (Table 8) were observed with this vector. This vector could be further modified to replace RNA-OUT with a different RNA selection marker, such as pMB1 RNAI or IncB RNAI.

Improved expression was observed when the gastrin eukaryotic transcription terminator was inserted into the spacer region (NTC9385R2a-O2-Gt versus NTC9385R2a-O2; Table 7). Collectively, these results demonstrate additional functionalities may be added to the spacer region without interfering with replicative minicircle performance.

Additional sequences that may be added to the spacer include bacterial selectable markers (e.g. RNA-OUT or RNAI; see Example 6), eukaryotic selectable markers, bacterial transcription terminators, eukaryotic transcription terminators (e.g. gastrin terminator), boundary elements, S/MARs, RNA Pol I or RNA Pol III expressed sequences or other functionalities. As well, additional sequences could be encoded within the intron, such as RNA Pol III transcription units expressing short hairpin RNA's or immunostimulatory RNAs such as those disclosed in Williams, J A Supra, 2008, included herein by reference.

Example 6: RNAI Regulated Vectors

Alternative RNA selection markers know in the art may be utilized in replicative minicircle vectors. For example, RNA-OUT (RNA-IN regulated chromosomal selection marker) may be replaced with the pMB 1 plasmid origin encoded RNAI (RNAII regulated chromosomal selection marker Grabherr and, Pfaffenzeller Supra,. 2006; Cranenburgh Supra, 2009), plasmid pMU720 origin encoded RNAI (SEQ ID NO: 35) that represses RNA II regulated targets (Wilson et al Supra, 1997), plasmid R1ParB locus Sok (Hok regulated chromosomal selection marker; Morsey Supra, 1999), F plasmid Flm locus FlmB (flmA regulated chromosomal selection marker; Morsey Supra, 1999) or other RNA selection markers described in the art. The use of alternative RNA selection markers to construct replicative minicircles was demonstrated here by substitution of RNA-OUT with the pMB1 plasmid origin encoded RNAI and assessing expression and splicing accuracy.

RNAI is present within the intron of the NTC9385P2 and NTC9385P2a vectors (FIG. 3; FIG. 4) and NTC9385P2(0.85) and NTC9385P2a(0.85) vectors (Example 7). The observed accurate splicing (Table 3) and robust expression (Table 4) of NTC9385P2 clones with RNAI in either orientation demonstrated that intronic pMB1 plasmid origin encoded RNAI expression is compatible with replicative minicircle function. The increased in vivo expression observed with NTC9385P2a-O1-muSEAP (FIG. 5) further demonstrates that intronic pMB1 plasmid origin encoded RNAI expression is compatible with replicative minicircle function. The observed accurate splicing (Table 8) and robust expression (Table 9) of NTC9385P2(0.85) clones with RNAI in either orientation demonstrated that intronic pMBl plasmid origin encoded RNAI expression is compatible with replicative minicircle function.

Nanoplasmid variants with the pMB 1 antisense RNA RNAI (SEQ ID NO:33) with promoter and terminator region (RNAI selectable marker: SEQ ID NO:34 flanked by DraIII-KpnI restriction sites for cloning as described previously for RNA-OUT) substituted for RNA-OUT were constructed as described in Example 2 and tested for expression to determine if alternative selection markers may be utilized in place of RNA-OUT. The results (Table 7) demonstrate alternative RNA selection markers may be substituted for RNA-OUT. Substitution of RNAI for RNA-OUT in the vector backbone spacer region (NTC9385Ra-RNAI-O1) or in the intron in either orientation (NTC9385R-RNAI-O1 and NTC9385R-RNAI-02) did not reduce expression relative to the corresponding RNA-OUT construct. To determine splicing accuracy, NTC9385R-RNAI-O1-EGFP and NTC9385R-RNAI-O2-EGFP were transfected into the A549 cell line and cytoplasmic RNA isolated from transfected A549 cells using the protein and RNA isolation system (PARIS kit, Ambion, Austin, Tex.) and quantified by A₂₆₀. Samples were DNase treated (DNA-free DNase; Ambion, Austin, Tex.) prior to reverse transcription using the Agpath-ID One step RT-PCR kit (Ambion, Austin, Tex.) with the EGFP transgene specific complementary strand primer EGFPR (FIG. 1). Intron splicing was determined by PCR amplification of the reverse transcribed cytoplasmic RNA with the EGFP5Rseq and CMVF5seq primers (FIG. 1). The resultant PCR product (a single band in each case) was determined by sequencing to be the correct spliced exon1-exon2 fragment (Table 8). This demonstrated that, like intronic RNA-OUT, intronic RNAI in either orientation is accurately removed by splicing and does not interfere with splicing accuracy. This further demonstrates that alternative RNA based selection markers may be substituted for RNA-OUT in the spacer region or the intron and that pMB 1 RNAI is a preferred RNA based selection marker for replicative minicircle vectors.

TABLE 7 High level expression with vectors with pMB1 RNAI encoded in the spacer region or intron A549 FU^(c) HEK293 FU^(c) Vector (all Spacer SR ^(d) (T = 48 (T = 48 EGFP) region ^(a) (bp) Intron ^(a) mean + SD) mean + SD) NTC8685 T-VA1- 1465 HR- β^(b)  8546 ± 1163 62068 ±1760  BH-P-AF- (1.00x) (1.00x) SV40 NTC9385R2- None 0 HR←AF R -T →- 15394 ± 683 30995 ± 4487 O2 β (1.80x) (0.50x) NTC9385R2a- BE 0 HR←AF R -T →- 11383 ± 253 36382 ± 1086 O2 β (1.33x) (0.59x) NTC9385R2a- TT -BE 73 HR←AF R -T →- 15076 ± 321 49289 ± 2672 O2-Gt β (1.76x) (0.79x) NTC9385R2b- AF→ 148 HR←R -T →-β  10721 ± 1039 42507 ± 5321 O2 (1.25x) (0.68x) NTC9385Ra- ←R - 466 HR-AF→- β  13929 ± 1291 56552 ± 2714 O1 dual AF→ (1.63x) (0.91x) NTC9385Ra- ←R - 466 HR-←AF- β 12543 ± 245 54379 ± 1244 O2 dual AF→ (1.47x) (0.89x) NTC9385Ra- ←R - 488 HR-AF→- β 15773 ± 238 55468 ± 6619 RNAI-O1 RNAI→ (1.85x) (0.89x) NTC9385R- ←R - 466 HR-← RNAI - β 14296 ± 287 60630 ± 2176 RNAI-O1 AF→ (1.67x) (0.98x) NTC9385R- ←R - 466 HR- RNAI→- β 12271 ± 466 60691 ± 6482 RNAI-O2 AF→ (1.44x) (0.98x) ^(a) trpA term = T; Gastrin (Gt) eukaryotic terminator = TT; HTLV-IR = HR; B globin 3′ acceptor site = β; RNA-OUT sucrose selection marker = AF; pUC origin RNAI antisense RNA = RNAI; pUC origin = P; R6K origin = R; ColE2 origin = C; CMV boundary element = BE; PAS-BH = BH; UP = upstream pUC plasmid derived DNA ^(b)HR β intron is 225 bp ^(c)EGFP plasmid DNA transfected with Lipofectamine 2000. Mean ± SD Fluorescence (FU) reported. ( ) Mean FU standardized to NTC8685 ^(d) Spacer Region (SR) size (bp) ss total bp of components between polyA and CMV or SV40 enhancer, and does not include the SV40 enhancer or BE..

TABLE 8 Accurate splicing with replicative minicircle vectors with pMB1 RNAI and minimal pUC origin encoded in the intron Predicted Actual EGFP % spliced spliced Cell RT-PCR EGFP exon size exon size Plasmid line (pg) mRNA (unspliced) (PCR) NTC9385R A549 87.5 ± 3.8 0.260% 279 (514) 279 ^(c) NTC9385R- A549 45.9 ± 3.0 0.142% 279 (667) 279 ^(c) RNAI-O1 NTC9385R- A549 43.1 ± 2.1 0.121% 279 (667) 279 ^(c) RNAI-O2 NTC9385R2-O2 A549 26.8 ± 2.2 0.094% 279 (974) 279 ^(c) NTC9385Ra-O2 A549 60.2 ± 6.1 0.198% 279 (648) 279 ^(c) NTC9385R2b-O2 A549 42.1 ± 1.2 0.148% 279 (819) 279 ^(c) NTC9385P2a-O1 A549 15.8 ± 1.3 0.054%  279 (1715) 279 ^(c) NTC9385P2a-O2 A549 11.6 ± 0.2 0.037%  279 (1715) 279 ^(a) NTC9385P2a A549 20.1 ± 1.6 0.085%  279 (1366) 279 ^(c) (0.85)-O1 NTC9385P2a A549  6.7 ± 0.5 0.027%  279 (1366) 279 ^(b) (0.85)-O2 NTC9385C- A549 0.003 0.000 No band No band C2x4-muSEAP (negative control) ^(a) Faint extra bands at 490 and 650 bp (previously observed with transfection of NTC9385P2a-O2 into A549; (see Table 3). Correct splice junction verified by DNA sequencing of PCR product. Faint band at 490 bp corresponds to mRNA with an additional pUC derived exon (see FIG. 3) ^(b) Very faint extra bands at 490 and 650 bp. Correct splice junction verified by DNA sequencing of PCR product. Faint band at 490 corresponds to mRNA with an additional pUC derived exon (see FIG. 3) ^(c) Correct splice junction verified by DNA sequencing of PCR product

The RNAI transcription unit (FIG. 10; SEQ ID NO: 34) may be substituted for the RNA-OUT selection marker (SEQ ID NO: 20) in any of the constructs described in Examples 2-6. Alternatively, the 108 by RNAI antisense repressor RNA (SEQ ID NO: 33) may be substituted for the 70 bp RNA-OUT antisense repressor RNA (SEQ ID NO: 21) retaining the flanking RNA-OUT transcription control sequences in any of the constructs described in Examples 2-6. RNAI regulated replicative minicircle vectors may be grown in RNAII-SacB regulated cell lines further expressing, as required, R6K, ColE2-P9, or ColE2 related rep protein. RNAII-SacB regulated cell lines may be made replacing the RNA-IN sequence in pCAH63-CAT RNA-IN-SacB (P5/6 6/6) with a RNAII target sequence as described in Williams, J A Supra, 2008 included herein by reference. Alternatively, RNAI regulated replicative minicircle vectors may be grown in any of the RNAII regulated chromosomal selection marker cell lines disclosed in Grabherr and, Pfaffenzeller Supra,. 2006 and Cranenburgh Supra, 2009. These cell lines would be modified for expression, as required, of R6K, ColE2-P9, or ColE2 related rep protein.

Another preferred RNA based selection marker, IncB plasmid RNAI (SEQ ID NO:35; SEQ ID NO:36), is shown in FIG. 11. A cell line for antibiotic free sucrose selection of IncB RNAI expressing plasmid vectors is created by modification of the genomically expressed RNA-IN-SacB cell lines for RNA-OUT plasmid propagation disclosed in Williams, J A Supra, 2008 by replacement of the 68 by RNA-IN regulator in a PstI-MamI restriction fragment with a 362 by PstI-MamI IncB RNAII regulator (SEQ ID NO:37).

Example 7: Minimal pUC origin replicative minicircles

Replicative minicircle vectors NTC8485P2 (0.85)-O1, NTC8485P2 (0.85)-O2, NTC9385P2a(0.85)-O1 and NTC9385P2a(0.85)-O2 containing the P_(min) pUC replication origin (SEQ ID NO: 38) and the RNA-OUT RNA selection marker (Bacterial region=SEQ ID NO: 39) were constructed as described in Example 2, and characterized for expression in HEK293 and A549 (Table 9) and splicing accuracy in A549 (Table 8) as described in Example 3. As with NTC9385P2a-O1 (Example 3) splicing was accurate with NTC9385P2a(0.85)-O1. Minor amounts of a cryptic P_(min) derived exon were detected with NTC9385P2a(0.85)-O2; the sequence of the cryptic exon matches the previously identified pUC derived cryptic exon observed with NTC9385P2a-O2, (Table 8; FIG. 3). Expression from both orientations was higher with NTC9385P2a(0.85)-O1 and NTC9385P2a(0.85)-O2 compared to NTC9385P2a-O1 and NTC9385P2a-O2 (Table 9) as well as NTC8485P2a(0.85)-O1 and NTC8485P2a(0.85)-O2 compared to NTC8485P2a-O1 and NTC8485P2a-O2 (Table 9). While not limiting the application of this invention, the higher expression with the intronic P_(min) replicative minicircles versus intronic pUC replicative minicircles may be due to smaller intron size or deletion of inhibitory sequences, such as the pUC origin nuclease sensitive site (FIG. 3). High yield manufacture was obtained with these intronic P_(min) pUC replication origin vectors (Table 6) with high quality plasmid surprisingly without detectable replication intermediates despite the close proximity of the P_(min) pUC replication origin and the CMV promoter enhancer.

TABLE 9 Robust expression with P2-(0.85) replicative minicircles A549 FU^(c) HEK293 FU^(c) (T = 48 (T = 48 Vector (EGFP) Spacer region ^(a) Intron ^(a) mean + SD) mean + SD) NTC8485 T← P-AF→SV40-BE HR -β 4311 ± 458 40236 ± 1851 (1x)   (1x)   NTC8485C2- T← P -AF→SV40-BE HR ←C AF→- β  5001 ± 2724  41334 ± 14098 O1 (1.16x) (1.03x) NTC8485C2- T← P -AF→SV40-BE HR ←AF C →- β 2962 ± 495 28849 ± 2421 O2 (0.69x) (0.72x) NTC8485R2- T← P -AF→SV40-BE HR←T-R AF→-β 2888 ± 180 29395 ± 1054 O1 (0.67x) (0.73x) NTC8485R2- T← P -AF→SV40-BE HR←AF R-T→-β 3187 ± 851 33044 ± 3515 O2 (0.74x) (0.82x) NTC8485P2- T← P -AF→SV40-BE HR← T-P-AF→-β 1143 ± 392 20775 ± 6777 O1 (0.27x) (0.52x) NTC8485P2- T← P -AF→SV40-BE HR←AF P -T →- 1500 ± 169 16575 ± 2483 O2 β (0.35x) (0.41x) NTC8485P2 T← P -AF→SV40-BE HR← T-P(0.85)- 1969 ± 591 31883 ± 2750 (0.85)-O1 AF→-β (0.46x) (0.79x) NTC8485P2- T← P -AF→SV40-BE HR←AF P(0.85) - 2171 ± 410 24733 ± 1417 (0.85)-O2 T →-B (0.50x) (0.61x) NTC9385P2a- None (BE) HR← T-P-AF→-β 4445 ± 217 26181 ± 1643 O1 (1.03x) (0.65x) NTC9385P2a- None (BE) HR←AF P -T →- 3457 ± 426 23829 ± 1514 O2 β (0.80x) (0.59x) NTC9385P2a None (BE) HR← T-P(0.85)- 6175 ± 258 38169 ± 2245 (0.85)-O1 AF→-β (1.43x) (0.95x) NTC9385P2a- None (BE) HR←AF P(0.85) - 6756 ± 583 35363 ± 3532 (0.85)-O2 T →-β (1.57x) (0.88x) NTC9385C2a- None (BE) HR← C AF→- B 5793 ± 820 36804 ± 6725 O1 (1.34x) (0.91 x) NTC9385R2a- None (BE) HR← T- R-AF→- 7498 ± 859 37595 ± 5497 O1 β (1.74x) (0.93x) NTC9385R2a- None (BE) HR←AF R-T →-β 5815 ± 456 36926 ± 2001 O2 (1.35x) (0.92x) ^(a) Prokaryotic terminator = T; HTLV-IR = HR; B globin 3′ acceptor site = β; RNA-OUT = AF; pUC origin = P; P_(min) minimalized pUC origin = P (0.85); R6Kγ origin = R; ColE2-P9 origin = C; Boundary element = BE; 2 × 72 bp repeat of SV40 enhancer = SV40 ^(c)EGFP plasmid DNA transfected with Lipofectamine 2000. Mean ± SD Fluorescence (FU) reported. ( ) Mean FU standardized to NTC8485

SUMMARY

While the above description contains many examples, these should not be construed as limitations on the scope of the invention, but rather should be viewed as an exemplification of preferred embodiments thereof. Many other variations are possible. For example, a replication origin and optionally a selectable marker may be inserted into the HTLV-I R-Rabbit β globin hybrid intron (SEQ ID NO:1) at any site between the 5′ splice acceptor and the 3′ acceptor branch site (FIG. 1) rather than the HpaI site. Alternatively, a replication origin and optionally a selectable marker may be inserted at two different sites within an intron between the 5′ splice acceptor and the 3′ acceptor branch site. Alternatively, a replication origin and a selectable marker may be inserted into two different introns, each insertion at any site between the 5′ splice acceptor and the 3′ acceptor branch site. Alternatively, a replication origin and a selectable marker may be inserted into alternative introns at any site between the 5′ splice acceptor and the 3′ acceptor branch site. A non limiting list of alternative introns for insertion of a bacterial region to create an intron encoded bacterial region of the invention are SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10. Additionally, the RNA-OUT selectable marker may be substituted with an alternative RNA-OUT sequence variant that functionally binds RNA-IN to repress expression. Likewise, the RNA-OUT promoter and/or terminator could be substituted with an alternative promoter and/or terminator. Further, an alternative RNA based selection marker could be substituted for RNA-OUT. This may be a plasmid borne nonsense suppressing tRNA that regulates a nonsense suppressible selectable chromosomal target as described by Crouzet and Soubrier Supra, 2005 included herein by reference. This may also be a plasmid borne antisense repressor RNA, a non limiting list included herein by reference includes pMB 1 plasmid origin encoded RNAI (SEQ ID NO: 33) that represses RNAII regulated targets (as described in Grabherr and, Pfaffenzeller Supra, 2006; Cranenburgh Supra, 2009), plasmid pMU720 origin encoded RNAI (SEQ ID NO: 35) that represses RNA II regulated targets (Wilson et al Supra, 1997) ParB locus Sok of plasmid RI that represses Hok regulated targets, Flm locus FhnB of F plasmid that represses fhnA regulated targets (Morsey Supra, 1999) or other antisense repressor RNAs known in the art. Likewise, the ColE2-P9 or R6K replication origin may be substituted with a CoIE2 related replication origin, and propagated in a strain expressing the CoIE2 related replication origin replication protein. Likewise, the ColE2-P9 or R6K Rep protein dependent origin may be substituted with an origin from one of the numerous alternative Rep protein dependent plasmids that are know in the art, for example the Rep protein dependent plasmids described in del Solar et al Supra, 1998 which is included herein by reference. Likewise, the vectors may encode a diversity of transgenes different from the examples provided herein, for example, antigen genes for a variety of pathogens, or therapeutic genes such as hypoxia inducible factor, keratinocyte growth factor, factor IX, factor VIII, etc, or RNA genes such as microRNAs or shRNA. Likewise, the vectors may utilize a diversity of RNA Pol H promoters different from the CMV promoter examples provided herein, for example, constitutive promoters such as the elongation factor 1 (EF 1) promoter, the chicken (3 -actin promoter, the -actin promoter from other species, the elongation factor-1 α (EF1 a) promoter, the phosphoglycerokinase (PGK) promoter, the Rous sarcoma virus (RSV) promoter, the human serum albumin (SA) promoter, the α-1 antitrypsin (AAT) promoter, the thyroxine binding globulin (TBG) promoter, the cytochrome P450 2E1 (CYP2E1) promoter, etc. The vectors may also utilize combination promoters such as the chicken β-actin/CMV enhancer (CAG) promoter, the human or murine CMV-derived enhancer elements combined with the elongation factor 1α(EF1a) promoters, CpG free versions of the human or murine CMV-derived enhancer elements combined with the elongation factor 1α(EF1α) promoters, the albumin promoter combined with an α-fetoprotein MERII enhancer, etc, or the diversity of tissue specific or inducible promoters know in the art such as the muscle specific promoters muscle creatine kinase (MCK), and C5-12 or the liver-specific promoter apolipoprotein A-I (ApoAl). Likewise the vectors could utilize a diversity of polyA signals known in the art, for example the bovine growth hormone, SV40 early or SV40 late polyA signals. The orientation of the various vector-encoded elements may be changed relative to each other.

The vectors may optionally contain additional functionalities, such as nuclear localizing sequences, and/or immunostimulatory RNA elements as disclosed in Williams, J A Supra, 2008 as part of the eukaryotic region or alternatively in introns or the spacer region.

Additional sequences may be added to the spacer, for example a eukaryotic selectable marker, bacterial transcription terminators, eukaryotic transcription terminators, boundary elements, S/MARs, RNA Pol I or RNA Pol III expressed sequences or other functionalities. For example, improved expression was observed when the gastrin eukaryotic transcription terminator was inserted into the spacer region (NTC9385R2a-O2-Gt versus NTC9385R2a-O2; Table 7). As well, additional sequences could be encoded within the intron, such as RNA Pol III transcription units expressing short hairpin RNA's or immunostimulatory RNAs such as those disclosed in Williams, J A Supra, 2008, included herein by reference.

Any eukaryotic expression vector can be converted into replicative minicircle expression vector of the invention by 1) Cloning a bacterial region into an intron; and 2) removing the existing vector backbone spacer region encoded bacterial region. If the vector does not contain an intron, an intron for insertion of the bacterial region can be added by standard cloning methodologies known in the art. More than one intron can be used to make a replicative minicircle, by cloning the replication origin into one intron and the selection marker into a second intron. Alternatively, the replication origin can be cloned into an intron, and the selection maker encoded within the spacer region created from excision of the existing vector backbone encoded bacterial region.

Thus, the reader will see that the improved replicative minicircle expression vectors of the invention provide for an approach to improve plasmid expression (i.e. through direct linkage of eukaryotic polyA and eukaryotic promoter) while dramatically improving manufacture compared to alterative short spacer region vectors such as minicircles. 

1-21. (canceled)
 22. A method of constructing a eukaryotic replicative pUC-free minicircle expression vector comprising: a. combining, under conditions so as to create a eukaryotic replicative pUC-free minicircle expression vector, i) a eukaryotic region encoding a gene of interest and comprising an intron and 5′ and 3′ ends, with ii) a spacer region linking the 5′ and 3′ ends of the eukaryotic region, said spacer region less than 500 basepairs in length, and with iii) a bacterial replication origin that is not the pUC origin and a RNA selectable marker positioned within said intron; and b. expressing said gene of interest in said pUC-free minicircle expression vector, wherein said gene of interest in said pUC-free minicircle expression vector is expressed at a higher level than a vector comprising a pUC origin encoding spacer region greater than 500 basepairs.
 23. The method of claim 22, wherein said RNA selectable marker is an RNA-IN regulating RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO:20, and SEQ ID NO:22.
 24. The method of claim 22, wherein said RNA selectable marker is selected from the group consisting of: an RNA-OUT selectable marker that encodes an RNA-IN regulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO: 21; an RNAI selectable marker that encodes an RNAII regulating RNAI RNA with at least 95% sequence identity to SEQ ID NO: 33; an IncB RNAI selectable marker encoding an RNAII regulating RNAI RNA with at least 95% sequence identity to SEQ ID NO:
 35. 25. The method of claim 22, wherein said bacterial replication origin is an R6K replication origin with at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 11, SEQ ID NO:
 12. 26. The method of claim 22, wherein said bacterial replication origin is an ColE2-P9 replication origin with at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:
 16. 27. The method of claim 22, wherein said eukaryotic region has at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 30, SEQ ID NO:
 31. 28. The method of claim 22, wherein said bacterial replication origin that is not the pUC origin and a RNA selectable marker comprises an R6K replication origin-RNA-OUT RNA selection marker with at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO:
 28. 29. The method of claim 22, wherein said bacterial replication origin that is not the pUC origin and a RNA selectable marker comprises an ColE2-P9 replication origin-RNA-OUT RNA selection marker with at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO:
 25. 30. The method of claim 22, wherein said intron is a functional variant with at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10.
 31. A method of constructing a eukaryotic replicative pUC-free minicircle expression vector comprising: a. combining, under conditions so as to create a eukaryotic replicative pUC-free minicircle expression vector, i) a eukaryotic region encoding a gene of interest and comprising an intron and 5′ and 3′ ends, with ii) a spacer region linking the 5′ and 3′ ends of the eukaryotic region sequences and a bacterial replication origin that is not the pUC origin and a RNA selectable marker, said spacer region less than 500 basepairs in length and said bacterial replication origin and said RNA selectable marker positioned within said intron; b. transforming said replicative minicircle expression vector into cells of an RNA selectable marker regulated bacterial cell line; c. isolating the resultant transformed bacterial cells by selection; and d. propagating the resultant transformed bacterial cells in culture under conditions such as to manufacture said vector in yields of greater than 100 mg vector per liter culture.
 32. The method of claim 31, wherein said RNA selectable marker is an RNA-IN regulating RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO:20, and SEQ ID NO:22.
 33. The method of claim 31, wherein said RNA selectable marker is selected from the group consisting of: an RNA-OUT selectable marker that encodes an RNA-IN regulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO: 21; an RNAI selectable marker that encodes an RNAII regulating RNAI RNA with at least 95% sequence identity to SEQ ID NO: 33; an IncB RNAI selectable marker encoding an RNAII regulating RNAI RNA with at least 95% sequence identity to SEQ ID NO:
 35. 34. The method of claim 31, wherein said bacterial replication origin is an R6K replication origin with at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 11, SEQ ID NO:
 12. 35. The method of claim 31, wherein said eukaryotic region has at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 30, SEQ ID NO:
 31. 36. A eukaryotic replicative pUC-free minicircle expression vector comprising i) a eukaryotic region sequence comprising an intron and 5′ and 3′ ends and ii) a spacer region of less than 500 basepairs in length linking the 5′ and 3′ ends of the eukaryotic region sequences and iii) a bacterial replication origin that is not the pUC origin and a RNA selectable marker positioned within said intron.
 37. The vector of claim 36, wherein said RNA selectable marker is an RNA-IN regulating RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO:20, and SEQ ID NO:22.
 38. The vector of claim 36, wherein said RNA selectable marker is selected from the group consisting of: an RNA-OUT selectable marker that encodes an RNA-IN regulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO: 21; an RNAI selectable marker that encodes an RNAII regulating RNAI RNA with at least 95% sequence identity to SEQ ID NO: 33; an IncB RNAI selectable marker encoding an RNAII regulating RNAI RNA with at least 95% sequence identity to SEQ ID NO:
 35. 39. The vector of claim 36, wherein said bacterial replication origin is an R6K replication origin with at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 11, SEQ ID NO:
 12. 40. The vector of claim 36, wherein said bacterial replication origin is an ColE2-P9 replication origin with at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:
 16. 41. The vector of claim 36, wherein said eukaryotic region has at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 30, SEQ ID NO:
 31. 